High-Density Lipoprotein in Metabolic Disorders and Beyond: An Exciting NewWorld Full of Challenges and Opportunities

High-density lipoprotein (HDL) is an enigmatic member of the plasma lipid and lipoprotein transport system, best known for its ability to promote the reverse cholesterol efflux and the unloading of excess cholesterol from peripheral tissues. More recently, data in experimental mice and humans suggest that HDL may play important novel roles in other physiological processes associated with various metabolic disorders. Important parameters in the HDL functions are its apolipoprotein and lipid content, further reinforcing the principle that HDL structure defines its functionality. Thus, based on current evidence, low levels of HDL-cholesterol (HDL-C) or dysfunctional HDL particles contribute to the development of metabolic diseases such as morbid obesity, type 2 diabetes mellitus, and nonalcoholic fatty liver disease. Interestingly, low levels of HDL-C and dysfunctional HDL particles are observed in patients with multiple myeloma and other types of cancer. Therefore, adjusting HDL-C levels within the optimal range and improving HDL particle functionality is expected to benefit such pathological conditions. The failure of previous clinical trials testing various HDL-C-raising pharmaceuticals does not preclude a significant role for HDL in the treatment of atherosclerosis and related metabolic disorders. Those trials were designed on the principle of “the more the better”, ignoring the U-shape relationship between HDL-C levels and morbidity and mortality. Thus, many of these pharmaceuticals should be retested in appropriately designed clinical trials. Novel gene-editing-based pharmaceuticals aiming at altering the apolipoprotein composition of HDL are expected to revolutionize the treatment strategies, improving the functionality of dysfunctional HDL

Study of the pleiotropic effects of apolipoprotein C3 in order to highlight innovative pharmacological interventions aiming at the functionality of HDL

Apolipoprotein C3 (APOC3) is mainly synthesized in the liver and intestine and its size is 8.8 kDa. It is predominantly distributed to lipoproteins characterized by high levels of apolipoprotein B (APOB-lipoproteins), such as chylomicrons, VLDL, IDL and LDL and approximately half of plasma APOC3 associates with HDL. Although there was a predominant view that APOC3 associates with HDL by simple binding to pre-existing particles, recent data support that biogenesis of APOC3-containing HDL (APOC3-HDL) requires Abca1. Moreover, APOC3-HDL contributes to plasma triglyceride homeostasis, by preventing APOC3 association with triglyceride-rich lipoproteins.Interestingly, APOC3-HDL also shows positive correlation with the morbidly obese phenotype. However, the roles of APOC3 in HDL functionality and adipose tissue metabolic activity remain unknown. In a clinical trial of morbidly obese subjects, we observed significant levels of APOC3-HDL particles in plasma immediately prior bariatric surgery, while six months after operation all HDL was mainly APOA1-HDL with only trace amounts of APOC3-HDL being present. This change in apolipoprotein content of HDL was associated with alterations in the antioxidant properties of HDL and plasma enzymatic activities of lecithin cholesterol acyltransferase (LCAT) and cholesterylester transfer protein (CETP). These clinical observations supported the interesting hypothesis that changes in APOC3 content of HDL associate with alterations in HDL particle functionality and overall body energy metabolism. To this date most of the studies have focused on the unique, broadly accepted, functional role of APOC3 in atherosclerosis, and more specifically in regulating plasma triglyceride levels by inhibiting Lpl activity and catabolism of triglyceride-rich lipoproteins. Based on this function, Apoc3 antisense oligonucelotides are currently in clinical trials for the treatment of hypertriglyceridemia. However, the roles of APOC3 in the function of HDL and metabolic activity of the adipose tissue remain unknown. Therefore, in the present study we investigated the direct effects of APOC3 expression on the structure and function of HDL, as well as on the metabolic activity of white (WAT) and brown adipose tissue (BAT).For this purpose C57BL/6 mice were infected with an adenovirus expressing human APOC3 or a control adenovirus AdGFP and five days post-infection blood and tissue samples were collected. HDL was then isolated and analyzed for its apolipoprotein and lipid content as well as for the structure and functionality of its particles. In addition, serum metabolomic analysis was performed by NMR and purified mitochondria from BAT and WAT were analyzed for Ucp1, Cytc and Cox4 protein levels as an indirect measure of their metabolic activity.Combined, our data show that APOC3 alters the apolipoprotein content of HDL, which in turn modulates its lipidome, resulting in significant changes in the structure and functionality of HDL. Apart from the effect of APOC3 on HDL, this apolipoprotein has a major effect on energy metabolism by activating the metabolic activity selectively in BAT.Η απολιποπρωτεΐνη C3 (APOC3) συντίθεται στο ήπαρ και το έντερο και έχει μέγεθος 8,8 kDa. Κατανέμεται κυρίως στις λιποπρωτεϊνες που χαρακτηρίζονται από υψηλή περιεκτικότητά σε απολιποπρωτεϊνη Β (APOΒ-λιποπρωτεΐνες), όπως τα χυλομικρά, η VLDL, η IDL και η LDL και περίπου το ήμισυ της APOC3 του πλάσματος σχετίζεται με την HDL. Αν και για χρόνια επικρατούσε η άποψη ότι η APOC3 σχετίζεται με την HDL λόγω απλής σύνδεσης με προϋπάρχοντα σωματίδια, πρόσφατα δεδομένα υποστηρίζουν ότι η βιογένεση της HDL που περιέχει APOC3 (APOC3-HDL) απαιτεί τη μεσολάβηση του μεταφορέα Abca1. Επιπλέον, η APOC3-HDL συμβάλλει στην ομοιόσταση των τριγλυκεριδίων του πλάσματος, εμποδίζοντας την σύνδεση της APOC3 με τις πλούσιες σε τριγλυκερίδια λιποπρωτεΐνες. Ενδιαφέρον είναι το γεγονός ότι η APOC3-HDL παρουσιάζει επίσης θετική συσχέτιση με τον παθολογικά παχύσαρκο φαινότυπο. Σε μια κλινική μελέτη, που περιλάμβανε ασθενείς με νοσογόνο παχυσαρκία (ΒΜΙ>50), παρατηρήσαμε σημαντικά επίπεδα HDL σωματιδίων πλούσιων σε APOC3 στο πλάσμα των ασθενών αμέσως πριν από τη βαριατρική χειρουργική επέμβαση, ενώ έξι μήνες μετά τα σωματίδια της HDL αποτελούνταν κυρίως από ΑΡΟΑΙ, ενώ η APOC3 ήταν ελάχιστη. Οι μεταβολές του απολιποπρωτεϊνικού περιεχομένου της HDL συσχετίστηκαν με μεταβολές στην ενζυμική δραστικότητα της LCAT και της πρωτεΐνης μεταφοράς χοληστερόλης (CETP), καθώς και στην αντιοξειδωτική ικανότητα της HDL. Οι μέχρι τώρα μελέτες έχουν εστιάσει στον μοναδικό, ευρύτερα αποδεκτό, λειτουργικό ρόλο της APOC3 που αφορά στην αθηροσκλήρωση και πιο συγκεκριμένα στη ρύθμιση των επιπέδων των τριγλυκεριδίων του πλάσματος μέσω της αναστολής της δραστικότητας της Lpl και του καταβολισμού των πλούσιων σε τριγλυκερίδια λιποπρωτεϊνών. Με βάση αυτή τη λειτουργία, ολιγονουκλεοτίδια με αντινοηματική ακολουθία (ASO) που στοχεύουν στη αποσιώπηση της έκφραση της APOC3 βρίσκονται σήμερα σε κλινικές δοκιμές για τη θεραπεία της υπερτριγλυκεριδαιμίας (HTG) και των σχετικών με αυτήν παθολογιών. Ωστόσο, ο ρόλος της APOC3 στη λειτουργικότητα της HDL και στη μεταβολική δραστηριότητα του λιπώδους ιστού παραμένει άγνωστος. Ως εκ τούτου, στην παρούσα μελέτη ερευνήσαμε τα άμεσα αποτελέσματα της έκφρασης της APOC3 στη δομή και τη λειτουργικόητα της HDL, καθώς και στη μεταβολική δραστηριότητα του λευκού (WAT) και του φαιού λιπώδους ιστού (BAT).Για το σκοπό αυτό χρησιμοποιήθηκαν C57BL/6 ποντίκια, στα οποία χορηγήθηκε ανασυνδυασμένος αδενοϊός που εκφράζει την ανθρώπινη APOC3 (AdGFP-APOC3) ή αδενοϊός ελέγχου AdGFP και συλλέχθηκαν δείγματα αίματος και ιστού πέντε ημέρες μετά τη χορήγηση. Στη συνέχεια απομονώθηκε η HDL και αναλύθηκε για το απολιποπρωτεϊνικό και το λιπιδικό της περιεχόμενο, καθώς και για τη δομή και τη λειτουργικότητα των σωματιδίων της. Επιπλέον, έγινε μεταβολομική ανάλυση του ορού με NMR και ανάλυση των επιπέδων των πρωτεϊνών Ucp1, Cytc και Cox4 στα μιτοχονδριακά κλάσματα που απομονώθηκαν από τους ιστούς ΒΑΤ και WAT, ως έμμεσοι δείκτες της μεταβολικής δραστηριότητάς τους.Συνδυασμένα, τα δεδομένα δείχνουν ότι η APOC3 μεταβάλλει το απολιποπρωτεϊνικό περιεχόμενο της HDL, το οποίο με τη σειρά του ρυθμίζει το λιπιδικό με αποτέλεσμα να προκαλούνται σημαντικές αλλαγές στη δομή και τη λειτουργικότητα της HDL. Εκτός όμως από την επίδραση της αυξημένης έκφρασης της APOC3 στην HDL, η απολιποπρωτεΐνη αυτή φάνηκε να παίζει σημαντικό ρόλο στην ενεργοποίηση του ενεργειακού μεταβολισμού, ενεργοποιώντας τη μεταβολική δραστηριότητα των μιτοχονδρίων του BAT

High-Density Lipoprotein in Metabolic Disorders and Beyond: An Exciting New World Full of Challenges and Opportunities

High-density lipoprotein (HDL) is an enigmatic member of the plasma lipid and lipoprotein transport system, best known for its ability to promote the reverse cholesterol efflux and the unloading of excess cholesterol from peripheral tissues. More recently, data in experimental mice and humans suggest that HDL may play important novel roles in other physiological processes associated with various metabolic disorders. Important parameters in the HDL functions are its apolipoprotein and lipid content, further reinforcing the principle that HDL structure defines its functionality. Thus, based on current evidence, low levels of HDL-cholesterol (HDL-C) or dysfunctional HDL particles contribute to the development of metabolic diseases such as morbid obesity, type 2 diabetes mellitus, and nonalcoholic fatty liver disease. Interestingly, low levels of HDL-C and dysfunctional HDL particles are observed in patients with multiple myeloma and other types of cancer. Therefore, adjusting HDL-C levels within the optimal range and improving HDL particle functionality is expected to benefit such pathological conditions. The failure of previous clinical trials testing various HDL-C-raising pharmaceuticals does not preclude a significant role for HDL in the treatment of atherosclerosis and related metabolic disorders. Those trials were designed on the principle of “the more the better”, ignoring the U-shape relationship between HDL-C levels and morbidity and mortality. Thus, many of these pharmaceuticals should be retested in appropriately designed clinical trials. Novel gene-editing-based pharmaceuticals aiming at altering the apolipoprotein composition of HDL are expected to revolutionize the treatment strategies, improving the functionality of dysfunctional HDL

Impact of apolipoprotein A1- or lecithin:cholesterol acyltransferase-deficiency on white adipose tissue metabolic activity and glucose homeostasis in mice

High density lipoprotein (HDL) has attracted the attention of biomedical community due to its well-documented role in atheroprotection. HDL has also been recently implicated in the regulation of islets of Langerhans secretory function and in the etiology of peripheral insulin sensitivity. Indeed, data from numerous studies strongly indicate that the functions of pancreatic \u3b2-cells, skeletal muscles and adipose tissue could benefit from improved HDL functionality. To better understand how changes in HDL structure may affect diet-induced obesity and type 2 diabetes we aimed at investigating the impact of Apoa1 or Lcat deficiency, two key proteins of peripheral HDL metabolic pathway, on these pathological conditions in mouse models. We report that universal deletion of apoa1 or lcat expression in mice fed western-type diet results in increased sensitivity to body-weight gain compared to control C57BL/6 group. These changes in mouse genome correlate with discrete effects on white adipose tissue (WAT) metabolic activation and plasma glucose homeostasis. Apoa1-deficiency results in reduced WAT mitochondrial non-shivering thermogenesis. Lcat-deficiency causes a concerted reduction in both WAT oxidative phosphorylation and non-shivering thermogenesis, rendering lcat 12/ 12 mice the most sensitive to weight gain out of the three strains tested, followed by apoa1 12/ 12 mice. Nevertheless, only apoa1 12/ 12 mice show disturbed plasma glucose homeostasis due to dysfunctional glucose-stimulated insulin secretion in pancreatic \u3b2-islets and insulin resistant skeletal muscles. Our analyses show that both apoa1 12/ 12 and lcat 12/ 12 mice fed high-fat diet have no measurable Apoa1 levels in their plasma, suggesting no direct involvement of Apoa1 in the observed phenotypic differences among groups

Distinct Roles of Apolipoproteins A1 and E in the Modulation of High-Density Lipoprotein Composition and Function

In addition to high-density lipoprotein cholesterol (HDL-C) levels, HDL quality also appears to be very important for atheroprotection. Analysis of various clinical paradigms suggests that the lipid and apolipoprotein composition of HDL defines its size, shape, and functions and may determine its beneficial effects on human health. Previously, we reported that like apolipoprotein A-I (Apoa1), apolipoprotein E (Apoe) is also capable of promoting the <i>de novo</i> biogenesis of HDL with the participation of ATP binding cassette A lipid transporter member 1 (Abca1) and plasma enzyme lecithin:cholesterol acyltransferase (Lcat), in a manner independent of a functional Apoa1. Here, we performed a comparative analysis of the functions of these HDL subpopulations. Specifically, Apoe and Apoa1 double-deficient (<i>Apoe</i>^{<i>–/–</i>} × <i>Apoa1</i>^{<i>–/–</i>}) mice were infected with <i>APOA1-</i> or <i>APOE3-</i>expressing adenoviruses, and APOA1-containing HDL (APOA1-HDL) and APOE3-containing HDL (APOE3-HDL), respectively, were isolated and analyzed by biochemical and physicochemical methods. Western blot and lipidomic analyses indicated significant differences in the apolipoprotein and lipid composition of the two HDL species. Moreover APOE3-HDL presented a markedly reduced antioxidant potential and Abcg1-mediated cholesterol efflux capacity. Surprisingly, APOE3-HDL but not APOA1-HDL attenuated LPS-induced production of TNFα in RAW264.7 cells, suggesting that the anti-inflammatory effects of APOA1 are dependent on APOE expression. Taken together, our data indicate that APOA1 and APOE3 recruit different apolipoproteins and lipids on the HDL particle, leading to structurally and functionally distinct HDL subpopulations. The distinct role of these two apolipoproteins in the modulation of HDL functionality may pave the way toward the development of novel pharmaceuticals that aim to improve HDL functionality