Metabolic regulation of the lysosomal cofactor bis(monoacylglycero)phosphate in mice

Abstract

Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid, is a phospholipid that promotes lipid sorting in late endosomes/lysosomes by activating lipid hydrolases and lipid transfer proteins. Changes in the cellular BMP content therefore reflect an altered metabolic activity of the endolysosomal system. Surprisingly, little is known about the physiological regulation of BMP. In this study, we investigated the effects of nutritional and metabolic factors on BMP profiles of whole tissues and parenchymal and nonparenchymal cells. Tissue samples were obtained from fed, fasted, 2 h refed, and insulin-treated mice, as well as from mice housed at 5°C, 22°C, or 30°C. These tissues exhibited distinct BMP profiles that were regulated by the nutritional state in a tissue-specific manner. Insulin treatment was not sufficient to mimic refeeding-induced changes in tissue BMP levels, indicating that BMP metabolism is regulated by other hormonal or nutritional factors. Tissue fractionation experiments revealed that fasting drastically elevates BMP levels in hepatocytes and pancreatic cells. Furthermore, we observed that the BMP content in brown adipose tissue strongly depends on housing temperatures. In conclusion, our observations suggest that BMP concentrations adapt to the metabolic state in a tissue- and cell-type-specific manner in mice. Drastic changes observed in hepatocytes, pancreatic cells, and brown adipocytes suggest that BMP plays a role in the functional adaption to nutrient starvation and ambient temperature. Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid, is a phospholipid that promotes lipid sorting in late endosomes/lysosomes by activating lipid hydrolases and lipid transfer proteins. Changes in the cellular BMP content therefore reflect an altered metabolic activity of the endolysosomal system. Surprisingly, little is known about the physiological regulation of BMP. In this study, we investigated the effects of nutritional and metabolic factors on BMP profiles of whole tissues and parenchymal and nonparenchymal cells. Tissue samples were obtained from fed, fasted, 2 h refed, and insulin-treated mice, as well as from mice housed at 5°C, 22°C, or 30°C. These tissues exhibited distinct BMP profiles that were regulated by the nutritional state in a tissue-specific manner. Insulin treatment was not sufficient to mimic refeeding-induced changes in tissue BMP levels, indicating that BMP metabolism is regulated by other hormonal or nutritional factors. Tissue fractionation experiments revealed that fasting drastically elevates BMP levels in hepatocytes and pancreatic cells. Furthermore, we observed that the BMP content in brown adipose tissue strongly depends on housing temperatures. In conclusion, our observations suggest that BMP concentrations adapt to the metabolic state in a tissue- and cell-type-specific manner in mice. Drastic changes observed in hepatocytes, pancreatic cells, and brown adipocytes suggest that BMP plays a role in the functional adaption to nutrient starvation and ambient temperature. brown adipose tissue bis(monoacyl­glycero)phosphate intraluminal vesicle nonparenchymal cell transcription factor EB white adipose tissue Lysosomes are the primary degradative organelles essential for the maintenance of metabolic homeostasis and health. Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid, is a phospholipid highly enriched in the intraluminal vesicles (ILVs) of late endosomes/lysosomes (1Kobayashi T. Beuchat M-H. Chevallier J. Makino A. Mayran N. Escola J-M. Lebrand C. Cosson P. Kobayashi T. Gruenberg J. Separation and characterization of late endosomal membrane domains.J. Biol. Chem. 2002; 277: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar) and plays a key role in lysosomal cargo sorting. BMP is a structural isomer of phosphatidylglycerol that harbors one FA at each of the two glycerol moieties. It has a unique sn-1-glycerophospho-sn-1′-glycerol stereoconformation, making it highly resistant to degradation by phospholipases of acidic organelles (2Tan H.H. Makino A. Sudesh K. Greimel P. Kobayashi T. Spectroscopic evidence for the unusual stereochemical configuration of an endosome-specific lipid.Angew. Chem. Int. Ed. Engl. 2012; 51: 533-535Crossref PubMed Scopus (34) Google Scholar). Because it is negatively charged at a lysosomal pH, it can act as a docking platform that recruits positively charged lipid hydrolases to ILVs, thereby facilitating the degradation of lipid cargo (3Schulze H. Kolter T. Sandhoff K. Principles of lysosomal membrane degradation: cellular topology and biochemistry of lysosomal lipid degradation.Biochim. Biophys. Acta. 2009; 1793: 674-683Crossref PubMed Scopus (178) Google Scholar). Additionally, BMP is an important cofactor in lysosomal cholesterol and sphingolipid metabolism through its interaction with cholesterol transport and sphingolipid activator proteins (4Locatelli-Hoops S. Remmel N. Klingenstein R. Breiden B. Rossocha M. Schoeniger M. Koenigs C. Saenger W. Sandhoff K. Saposin A mobilizes lipids from low cholesterol and high bis(monoacylglycerol)phosphate-containing membranes: patient variant Saposin A lacks lipid extraction capacity.J. Biol. Chem. 2006; 281: 32451-32460Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 5Enkavi G. Mikkolainen H. Güngör B. Ikonen E. Vattulainen I. Concerted regulation of npc2 binding to endosomal/lysosomal membranes by bis(monoacylglycero)phosphate and sphingomyelin.PLOS Comput. Biol. 2017; 13: e1005831Crossref PubMed Scopus (22) Google Scholar). BMP is considered on the basis of these versatile functions to be a key activator of lipid sorting and digestion (6Gallala H.D. Sandhoff K. Biological function of the cellular lipid BMP-BMP as a key activator for cholesterol sorting and membrane digestion.Neurochem. Res. 2011; 36: 1594-1600Crossref PubMed Scopus (77) Google Scholar). Recent data also suggest that BMP is a cofactor for heat shock protein 70, a chaperone that promotes cell survival by inhibiting lysosomal membrane permeabilization (7Kirkegaard T. Roth A.G. Petersen N.H.T. Mahalka A.K. Olsen O.D. Moilanen I. Zylicz A. Knudsen J. Sandhoff K. Arenz C. et al.Hsp70 stabilizes lysosomes and reverts Niemann–Pick disease-associated lysosomal pathology.Nature. 2010; 463: 549-553Crossref PubMed Scopus (368) Google Scholar). Changes in the cellular BMP content reflect ILV formation and degradation in acidic organelles and therefore indicate an altered metabolic activity of endolysosomal compartments. Little is known about the physiological regulation of BMP. However, under pathophysiological conditions, BMP concentrations are increased in numerous lysosomal storage disorders (8Hullin-Matsuda F. Kawasaki K. Delton-Vandenbroucke I. Xu Y. Nishijima M. Lagarde M. Schlame M. Kobayashi T. De novo biosynthesis of the late endosome lipid, bis(monoacylglycero)phosphate.J. Lipid Res. 2007; 48: 1997-2008Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) that can be caused by mutations in genes encoding lysosomal enzymes or as a side effect of drugs characterized by hydrophobic ring structures and side chains with a cationic amine group. These “cationic amphiphilic drugs” comprise many pharmacologic agents, including antibiotics, antidepressants, antiarrhythmics, cholesterol-lowering agents, and others (9Halliwell W.H. Cationic amphiphilic drug-induced phospholipidosis.Toxicol. Pathol. 1997; 25: 53-60Crossref PubMed Scopus (304) Google Scholar). They accumulate in lysosomes and disturb lipid degradation, possibly by interacting with negatively charged BMP (6Gallala H.D. Sandhoff K. Biological function of the cellular lipid BMP-BMP as a key activator for cholesterol sorting and membrane digestion.Neurochem. Res. 2011; 36: 1594-1600Crossref PubMed Scopus (77) Google Scholar). Genetic as well as drug-induced lysosomal storage disorders are associated with increased BMP concentrations in tissues and in the circulation, which is utilized in clinical practice as a biomarker for the diagnosis of these disorders (10Liu N. Tengstrand E.A. Chourb L. Hsieh F.Y. Di-22:6-bis(monoacylglycerol)phosphate: a clinical biomarker of drug-induced phospholipidosis for drug development and safety assessment.Toxicol. Appl. Pharmacol. 2014; 279: 467-476Crossref PubMed Scopus (51) Google Scholar). We recently demonstrated that high-fat-induced metabolic disease is also associated with elevated hepatic and circulating BMP concentrations (11Grabner G.F. Fawzy N. Pribasnig M. Trieb M. Taschler U. Holzer M. Schweiger M. Wolinski H. Kolb D. Horvath A. et al.Metabolic disease and ABHD6 alter the circulating bis(monoacylglycerol)phosphate profile in mice and humans.J. Lipid Res. 2019; 60: 1020-1031Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Furthermore, published data demonstrate that excessive dietary lipids cause lysosomal lipid storage and BMP accumulation in the kidney (12Rampanelli E. Ochodnicky P. Vissers J.P.C. Butter L.M. Claessen N. Calcagni A. Kors L. Gethings L.A. Bakker S.J.L. de Borst M.H. et al.Excessive dietary lipid intake provokes an acquired form of lysosomal lipid storage disease in the kidney.J. Pathol. 2018; 246: 470-484Crossref PubMed Scopus (24) Google Scholar). These changes possibly reflect adaptations to the dietary lipid overload causing an enhanced lipid flux through lysosomes, and on the basis of its versatile function in cargo sorting, it can be assumed that BMP is synthesized to facilitate metabolite flux through the disturbed lysosomal compartment. Accordingly, it was demonstrated that increased BMP levels induce cholesterol clearance in Niemann-Pick C disease in vitro and in vivo (13Moreau D. Vacca F. Vossio S. Scott C. Colaco A. Paz Montoya J. Ferguson C. Damme M. Moniatte M. Parton R.G. et al.Drug‐induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann–Pick type C cells and mice.EMBO Rep. 2019; 20: e47055Crossref PubMed Scopus (19) Google Scholar). Niemann-Pick C disease is characterized by the accumulation of unesterified cholesterol in late endosomal/lysosomal compartments (14Wheeler S. Sillence D.J. Niemann–Pick type C disease: cellular pathology and pharmacotherapy.J. Neurochem. 2019; (Epub ahead of print. October 14, 2019; doi:10.1111/jnc.14895.)PubMed Google Scholar) and is caused by mutations in the cholesterol transport proteins NPC1 or NPC2. Interestingly, increased BMP levels counteract cholesterol accumulation in cells lacking functional NPC1 but not NPC2, indicating that the interaction of BMP with NPC2 is essential for promoting cholesterol clearance (15McCauliff L.A. Langan A. Li R. Ilnytska O. Bose D. Waghalter M. Lai K. Kahn P.C. Storch J. Intracellular cholesterol trafficking is dependent upon NPC2 interaction with lysobisphosphatidic acid.eLife. 2019; 8: 1-31Crossref Scopus (30) Google Scholar). Despite its importance in cellular physiology and its association with human disease, the molecular basics of BMP metabolism and its regulation remain elusive. To obtain fundamental insights into the physiological regulation of BMP, we studied the effects of nutritional and metabolic factors on BMP content and FA profiles. Drastic changes upon fasting and cold exposure implicate that BMP is important in the functional adaptation to changing metabolic conditions. Animal experiments were approved by the Austrian Federal Ministry for Science, Research, and Economy and the ethics committee of the University of Graz and conducted in compliance with the Council of Europe Convention. C57Bl6/J mice were bred and maintained on a regular light-dark cycle (14 h light, 10 h dark) at 22 ± 1°C in a specific pathogen-free barrier facility. A standard laboratory chow diet (R/M-H Extrudate; Ssniff Spezialdiäten GmbH, Soest, Germany) and drinking water were provided ad libitum unless otherwise indicated. Age-matched male mice were used for all studies. Primary mouse hepatocytes and nonparenchymal cells (NPCs) were isolated as described previously with some modifications (16Taschler U. Schreiber R. Chitraju C. Grabner G.F. Romauch M. Wolinski H. Haemmerle G. Breinbauer R. Zechner R. Lass A. et al.Adipose triglyceride lipase is involved in the mobilization of triglyceride and retinoid stores of hepatic stellate Biophys. Acta. PubMed Scopus Google Scholar). 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A. et of in and PubMed Scopus Google Scholar). plays a key role in the regulation of and the nutritional regulation of BMP in the to the nutritional state the BMP content of pancreatic and cells. was associated with a increase of BMP in pancreatic and cells, indicating the formation of in acidic functional of BMP accumulation in pancreatic cells on its important role in cargo sorting, it can be that the degradation, or of and we that is the tissues with the BMP into parenchymal cells and a that the of BMP is in brown indicating a highly lysosomal system. is the for It in the membrane of which the from to heat A. Y. of in brown 2012; Full Text Full Text PDF PubMed Scopus Google Scholar). To its function in on a with in the form of B. J. adipose function and physiological PubMed Scopus Google Scholar) that can be in two lipase a high and and that are the FA J. L. adipose tissue and lipid 2018; PubMed Scopus Google Scholar). brown adipocytes can whole A. R. H. H. K. H. C. et adipose tissue activity triglyceride 2011; PubMed Scopus Google Scholar). the increased and degradation of also an adaption of the endosomal/lysosomal system. We observed that and BMP content strongly with the housing temperature. increase upon cold adaptation and at that BMP as activator of lysosomal lipid hydrolases (6Gallala H.D. Sandhoff K. Biological function of the cellular lipid BMP-BMP as a key activator for cholesterol sorting and membrane digestion.Neurochem. Res. 2011; 36: 1594-1600Crossref PubMed Scopus (77) Google the increased BMP content lysosomal degradation of and the of for and observed changes in tissue BMP content in to fasting or cold exposure on enzymes de novo degradation, of BMP. We recently that is involved in the degradation of BMP (11Grabner G.F. Fawzy N. Pribasnig M. Trieb M. Taschler U. Holzer M. Schweiger M. Wolinski H. Kolb D. Horvath A. et al.Metabolic disease and ABHD6 alter the circulating bis(monoacylglycerol)phosphate profile in mice and humans.J. Lipid Res. 2019; 60: 1020-1031Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, I. Grabner G.F. Taschler U. O. B. C. L. J. et the late endosomal/lysosomal lipid bis(monoacylglycero)phosphate.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). BMP it is well that phosphatidylglycerol is a of BMP (8Hullin-Matsuda F. Kawasaki K. Delton-Vandenbroucke I. Xu Y. Nishijima M. Lagarde M. Schlame M. Kobayashi T. De novo biosynthesis of the late endosome lipid, bis(monoacylglycero)phosphate.J. Lipid Res. 2007; 48: 1997-2008Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, J. F. Makino A. S. A. Kobayashi T. Lagarde M. Delton-Vandenbroucke I. of bis(monoacylglycero)phosphate content in by high with Lipid Res. 2009; Full Text Full Text PDF PubMed Scopus (34) Google Scholar). the of enzymes BMP from phosphatidylglycerol elusive. It is to on the basis of the in BMP FA tissues that or BMP in tissues high for distinct FA the important function of BMP in lysosomes, of the metabolic BMP and degradation important insights into the regulation of lysosomal In conclusion, our observations demonstrate that tissues BMP profiles that adapt to the nutritional and metabolic state in a tissue- and cell-type-specific manner. changes observed in hepatocytes, brown and pancreatic cells suggest that BMP plays a role in the functional adaption to nutrient and ambient temperatures. the described in the are in the published S. for

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