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• W2895084776 abstract "Sphingolipids, including ceramide, SM, and hexosylceramide (HxCer), are carried in the plasma by lipoproteins. They are possible markers of metabolic diseases, but little is known about their control. We previously showed that microsomal triglyceride transfer protein (MTP) is critical to determine plasma ceramide and SM, but not HxCer, levels. In human plasma and mouse models, we examined possible HxCer-modulating pathways, including the role of ABCA1 in determining sphingolipid plasma concentrations. Compared with control samples, plasma from patients with Tangier disease (deficient in ABCA1) had significantly lower HxCer (−69%) and SM (−40%) levels. Similarly, mice deficient in hepatic and intestinal ABCA1 had significantly reduced HxCer (−79%) and SM (−85%) levels. Tissue-specific ablation studies revealed that hepatic ABCA1 determines plasma HxCer and SM levels; that ablation of MTP and ABCA1 in the liver and intestine reduces plasma HxCer, SM, and ceramide levels; and that hepatic and intestinal MTP contribute to plasma ceramide levels, whereas only hepatic MTP modulates plasma SM levels. These results identify the contribution of ABCA1 to plasma SM and HxCer levels and suggest that MTP and ABCA1 are critical determinants of plasma sphingolipid levels. Sphingolipids, including ceramide, SM, and hexosylceramide (HxCer), are carried in the plasma by lipoproteins. They are possible markers of metabolic diseases, but little is known about their control. We previously showed that microsomal triglyceride transfer protein (MTP) is critical to determine plasma ceramide and SM, but not HxCer, levels. In human plasma and mouse models, we examined possible HxCer-modulating pathways, including the role of ABCA1 in determining sphingolipid plasma concentrations. Compared with control samples, plasma from patients with Tangier disease (deficient in ABCA1) had significantly lower HxCer (−69%) and SM (−40%) levels. Similarly, mice deficient in hepatic and intestinal ABCA1 had significantly reduced HxCer (−79%) and SM (−85%) levels. Tissue-specific ablation studies revealed that hepatic ABCA1 determines plasma HxCer and SM levels; that ablation of MTP and ABCA1 in the liver and intestine reduces plasma HxCer, SM, and ceramide levels; and that hepatic and intestinal MTP contribute to plasma ceramide levels, whereas only hepatic MTP modulates plasma SM levels. These results identify the contribution of ABCA1 to plasma SM and HxCer levels and suggest that MTP and ABCA1 are critical determinants of plasma sphingolipid levels. Low density apoB-containing lipoproteins (B-lps) and HDLs carry the bulk of lipids in the blood circulation. These lipoproteins carry triacylglycerols, phospholipids, cholesterol, and sphingolipids (1Hammad S.M. Pierce J.S. Soodavar F. Smith K.L. Al Gadban M.M. Rembiesa B. Klein R.L. Hannun Y.A. Bielawski J. Bielawska A. Blood sphingolipidomics in healthy humans: impact of sample collection methodology.J. Lipid Res. 2010; 51: 3074-3087Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 2Hammad S.M. Blood sphingolipids in homeostasis and pathobiology.Adv. Exp. Med. Biol. 2011; 721: 57-66Crossref PubMed Scopus (39) Google Scholar, 3Hammad S.M. Al Gadban M.M. Semler A.J. Klein R.L. Sphingosine 1-phosphate distribution in human plasma: associations with lipid profiles.J. Lipids. 2012; 2012: 180705Crossref PubMed Google Scholar, 4Wiesner P. Leidl K. Boettcher A. Schmitz G. Liebisch G. Lipid profiling of FPLC-separated lipoprotein fractions by electrospray ionization tandem mass spectrometry.J. Lipid Res. 2009; 50: 574-585Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 5Dawson G. Kruski A.W. Scanu A.M. Distribution of glycosphingolipids in the serum lipoproteins of normal human subjects and patients with hypo- and hyperlipidemias.J. Lipid Res. 1976; 17: 125-131Abstract Full Text PDF PubMed Google Scholar). The major sphingolipids in the plasma are SM, ceramide (Cer), hexosylceramide (HxCer), and lactosylceramide (LactCer). Sphingolipids consist of a common 18-carbon amino-alcohol backbone, sphingosine. These molecules are structurally diverse and regulate significant physiologic functions associated with several metabolic diseases such as insulin resistance, progression of nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH), atherosclerosis, and coronary artery disease (6Iqbal J. Walsh M.T. Hammad S.M. Hussain M.M. Sphingolipids and lipoproteins in health and metabolic disorders.Trends Endocrinol. Metab. 2017; 28: 506-518Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 7Hussain M.M. Jin W. Jiang X.C. Mechanisms involved in cellular ceramide homeostasis.Nutr. Metab. (Lond.). 2012; 9: 71Crossref PubMed Scopus (25) Google Scholar, 8Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2440) Google Scholar, 9Futerman A.H. Hannun Y.A. The complex life of simple sphingolipids.EMBO Rep. 2004; 5: 777-782Crossref PubMed Scopus (535) Google Scholar, 10Gault C.R. Obeid L.M. Hannun Y.A. An overview of sphingolipid metabolism: from synthesis to breakdown.Adv. Exp. Med. Biol. 2010; 688: 1-23Crossref PubMed Scopus (664) Google Scholar, 11Lucki N.C. Sewer M.B. Nuclear sphingolipid metabolism.Annu. Rev. Physiol. 2012; 74: 131-151Crossref PubMed Scopus (54) Google Scholar, 12Sabourdy F. Kedjouar B. Sorli S.C. Colie S. Milhas D. Salma Y. Levade T. Functions of sphingolipid metabolism in mammals–lessons from genetic defects.Biochim. Biophys. Acta. 2008; 1781: 145-183Crossref PubMed Scopus (60) Google Scholar, 13Hannun Y.A. Obeid L.M. Sphingolipids and their metabolism in physiology and disease.Nat. Rev. Mol. Cell Biol. 2018; 19: 175-191Crossref PubMed Scopus (808) Google Scholar). Earlier, we showed that microsomal triglyceride transfer protein (MTP) is a critical determinant of plasma Cer levels and partially contributes to plasma SM by investigating sphingolipid concentrations in the plasma of MTP-deficient abetalipoproteinemia subjects as well as in liver- and intestine-specific MTP-deficient mice (14Iqbal J. Walsh M.T. Hammad S.M. Cuchel M. Tarugi P. Hegele R.A. Davidson N.O. Rader D.J. Klein R.L. Hussain M.M. Microsomal triglycerdie transfer protein transfers and determines plasma concentrations of ceramide and sphingomyelin but not glycosylceramide.J. Biol. Chem. 2015; 290: 25863-25875Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). MTP deficiency in humans and mice was associated with ∼80% reductions in plasma Cer levels and ∼40% reduction in plasma SM levels. Mechanistic studies showed that MTP does not play a role in sphingolipid biosynthesis but is important for sphingolipid secretion. Furthermore, the data suggested that MTP might be involved in the intracellular transfer of Cer and SM to B-lps to facilitate their secretion. These studies also showed that MTP deficiency had no effect on plasma HxCer levels. This was surprising, as most, if not all, of these sphingolipids are present in B-lps (1Hammad S.M. Pierce J.S. Soodavar F. Smith K.L. Al Gadban M.M. Rembiesa B. Klein R.L. Hannun Y.A. Bielawski J. Bielawska A. Blood sphingolipidomics in healthy humans: impact of sample collection methodology.J. Lipid Res. 2010; 51: 3074-3087Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 15Chatterjee S. Sphingolipids in atherosclerosis and vascular biology.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1523-1533Crossref PubMed Scopus (221) Google Scholar). Here, we examined pathways that might modulate plasma HxCer levels. We hypothesized that, instead of B-lps, HDL might be involved in bringing these glycosphingolipids to the plasma compartment. Plasma HxCer species have been identified as possible biomarkers of disease progression. It has been shown that plasma HxCer species were high in chronic hepatitis C virus patients with severe fibrosis and hepatic necroinflammation (16Li J.F. Qu F. Zheng S.J. Ren J.Y. Wu H.L. Liu M. Liu H. Ren F. Chen Y. Zhang J.L. et al.Plasma sphingolipids as potential indicators of hepatic necroinflammation in patients with chronic hepatitis C and normal alanine aminotransferase level.PLoS One. 2014; 9: e95095Crossref PubMed Scopus (0) Google Scholar, 17Li J.F. Qu F. Zheng S.J. Ren F. Wu H.L. Liu M. Ren J.Y. Chen Y. Duan Z.P. Zhang J.L. Plasma sphingolipids: potential biomarkers for severe hepatic fibrosis in chronic hepatitis C.Mol. Med. Rep. 2015; 12: 323-330Crossref PubMed Scopus (13) Google Scholar). Furthermore, HxCer levels were closely associated with severe fibrosis in these patients following adjustment for several confounding variables in multivariate analysis (17Li J.F. Qu F. Zheng S.J. Ren F. Wu H.L. Liu M. Ren J.Y. Chen Y. Duan Z.P. Zhang J.L. Plasma sphingolipids: potential biomarkers for severe hepatic fibrosis in chronic hepatitis C.Mol. Med. Rep. 2015; 12: 323-330Crossref PubMed Scopus (13) Google Scholar). It has been suggested that HxCer (d18:1/12:0) could be a potential diagnostic marker for severe hepatic steatosis. The reasons for high plasma HxCer in fibrosis are not known (17Li J.F. Qu F. Zheng S.J. Ren F. Wu H.L. Liu M. Ren J.Y. Chen Y. Duan Z.P. Zhang J.L. Plasma sphingolipids: potential biomarkers for severe hepatic fibrosis in chronic hepatitis C.Mol. Med. Rep. 2015; 12: 323-330Crossref PubMed Scopus (13) Google Scholar). HxCer (16:1) has also been suggested to be a possible biomarker for disease progression in relapsing multiple sclerosis patients (18Checa A. Khademi M. Sar D.G. Haeggstrom J.Z. Lundberg J.O. Piehl F. Olsson T. Wheelock C.E. Hexosylceramides as intrathecal markers of worsening disability in multiple sclerosis.Mult. Scler. 2015; 21: 1271-1279Crossref PubMed Scopus (33) Google Scholar) and in drug-induced hepatic phospholipidosis (19Saito K. Maekawa K. Ishikawa M. Senoo Y. Urata M. Murayama M. Nakatsu N. Yamada H. Saito Y. Glucosylceramide and lysophosphatidylcholines as potential blood biomarkers for drug-induced hepatic phospholipidosis.Toxicol. Sci. 2014; 141: 377-386Crossref PubMed Scopus (25) Google Scholar). Plasma membrane-anchored ABCA1 actively participates in concert with extracellular acceptors (mainly apoAI) to generate HDL (20Westerterp M. Bochem A.E. Yvan-Charvet L. Murphy A.J. Wang N. Tall A.R. ATP-binding cassette transporters, atherosclerosis, and inflammation.Circ. Res. 2014; 114: 157-170Crossref PubMed Scopus (194) Google Scholar, 21Tall A.R. Role of ABCA1 in cellular cholesterol efflux and reverse cholesterol transport.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 710-711Crossref PubMed Scopus (45) Google Scholar, 22Yvan-Charvet L. Wang N. Tall A.R. Role of HDL, ABCA1, and ABCG1 transporters in cholesterol efflux and immune responses.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 139-143Crossref PubMed Scopus (477) Google Scholar, 23Schmitz G. Langmann T. Transcriptional regulatory networks in lipid metabolism control ABCA1 expression.Biochim. Biophys. Acta. 2005; 1735: 1-19Crossref PubMed Scopus (181) Google Scholar, 24Phillips M.C. Molecular mechanisms of cellular cholesterol efflux.J. Biol. Chem. 2014; 289: 24020-24029Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). This process is regulated by liver X receptors (LXRs) (25Schulman I.G. Liver X receptors link lipid metabolism and inflammation.FEBS Lett. 2017; 591: 2978-2991Crossref PubMed Scopus (116) Google Scholar, 26Zelcer N. Tontonoz P. Liver X receptors as integrators of metabolic and inflammatory signaling.J. Clin. Invest. 2006; 116: 607-614Crossref PubMed Scopus (766) Google Scholar). ABCA1 deficiency in Tangier disease significantly lowers plasma HDL and increases tissue sterols and premature coronary atherosclerosis in some individuals (27Assmann G. Von Eckardstein A. Brewer Jr., H.B. Familial high density lipoprotein deficiency: Tangier disease..The Metabolic and Molecular Bases of Inherited Disease. 2005; : 2053-2072Google Scholar, 28Parks J.S. Chung S. Shelness G.S. Hepatic ABC transporters and triglyceride metabolism.Curr. Opin. Lipidol. 2012; 23: 196-200Crossref PubMed Scopus (28) Google Scholar). ABCA1 deficiency in mice reduces plasma HDL and increases atherosclerosis (29Francone O.L. Aiello R.J. ABCA1: regulation, function and relationship to atherosclerosis.Curr. Opin. Investig. Drugs. 2002; 3: 415-419PubMed Google Scholar). Tissue-specific ablation studies in mice showed that hepatic and intestinal ABCA1 contribute to ∼70% and 30% of plasma HDL cholesterol, respectively (30Timmins J.M. Lee J.Y. Boudyguina E. Kluckman K.D. Brunham L.R. Mulya A. Gebre A.K. Coutinho J.M. Colvin P.L. Smith T.L. et al.Targeted inactivation of hepatic Abca1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism of apoA-I.J. Clin. Invest. 2005; 115: 1333-1342Crossref PubMed Scopus (423) Google Scholar, 31Brunham L.R. Kruit J.K. Iqbal J. Fievet C. Timmins J.A. Pape T.D. Coburn B.A. Bissada N. Staels B. Groen A.K. et al.Intestinal ABCA1 directly contributes to HDL biogenesis in vivo.J. Clin. Invest. 2006; 116: 1052-1062Crossref PubMed Scopus (419) Google Scholar). The effect of ABCA1 deficiency on plasma sphingolipids is unknown. Studies presented here show that ABCA1 is critical for modulating plasma HxCer and SM levels in humans and mice. The [3H]dihydrosphingosine (60 Ci/mmol) was purchased from American Radiolabeled Chemicals, Inc. C6-NBD-Cer (catalog no. 6224) was purchased from Setareh Biotech, and NBD-SM (catalog no. 229573) was from Avanti Polar Lipids. Human plasma samples from heterozygous abetalipoproteinemia (ABL, normal, n = 4) were treated as normal controls, as abetalipoproteinemia is an autosomal recessive disorder, and obligate heterozygous parents of these subjects have normal plasma lipids levels (32Berriot-Varoqueaux N. Aggerbeck L.P. Samson-Bouma M. Wetterau J.R. The role of the microsomal triglyceride transfer protein in abetalipoproteinemia.Annu. Rev. Nutr. 2000; 20: 663-697Crossref PubMed Scopus (264) Google Scholar, 33Walsh M.T. Hussain M.M. Targeting microsomal triglyceride transfer protein and lipoprotein assembly to treat homozygous familial hypercholesterolemia.Crit. Rev. Clin. Lab. Sci. 2017; 54: 26-48Crossref PubMed Scopus (23) Google Scholar). The same values have been used as controls in our previous study (14Iqbal J. Walsh M.T. Hammad S.M. Cuchel M. Tarugi P. Hegele R.A. Davidson N.O. Rader D.J. Klein R.L. Hussain M.M. Microsomal triglycerdie transfer protein transfers and determines plasma concentrations of ceramide and sphingomyelin but not glycosylceramide.J. Biol. Chem. 2015; 290: 25863-25875Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Heterozygous (HE; n = 5) and homozygous (HO; n = 6) Tangier subjects were collected at the University of Pennsylvania, after obtaining approvals by the university's Institutional Review Board and consents from subjects and/or parents. Frozen samples were shipped to SUNY Downstate Medical Center, where different analyses were performed. Additionally, appropriate aliquots of these samples were shipped from SUNY Downstate to the Lipidomics Shared Resource at the Medical University of South Carolina for sphingolipid analyses using LC/MS/MS as described previously (1Hammad S.M. Pierce J.S. Soodavar F. Smith K.L. Al Gadban M.M. Rembiesa B. Klein R.L. Hannun Y.A. Bielawski J. Bielawska A. Blood sphingolipidomics in healthy humans: impact of sample collection methodology.J. Lipid Res. 2010; 51: 3074-3087Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 14Iqbal J. Walsh M.T. Hammad S.M. Cuchel M. Tarugi P. Hegele R.A. Davidson N.O. Rader D.J. Klein R.L. Hussain M.M. Microsomal triglycerdie transfer protein transfers and determines plasma concentrations of ceramide and sphingomyelin but not glycosylceramide.J. Biol. Chem. 2015; 290: 25863-25875Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Chow-fed male, 10-week-old mice on a C57BL/6J background were used in these studies. Abca1 floxed (Abca1f/f) mice (30Timmins J.M. Lee J.Y. Boudyguina E. Kluckman K.D. Brunham L.R. Mulya A. Gebre A.K. Coutinho J.M. Colvin P.L. Smith T.L. et al.Targeted inactivation of hepatic Abca1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism of apoA-I.J. Clin. Invest. 2005; 115: 1333-1342Crossref PubMed Scopus (423) Google Scholar) were crossed with ERT2-villin-Cre mice to obtain ERT2-villin-Cre;Abca1f/f mice as described previously (34Iqbal J. Parks J.S. Hussain M.M. Lipid absorption defects in intestine-specific microsomal triglyceride transfer protein and ATP-binding cassette transporter A1-deficient mice.J. Biol. Chem. 2013; 288: 30432-30444Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). To generate liver- and intestine-specific ABCA1 ablated (L,I-Abca1−/−) mice, 10-week-old ERT2-villin-Cre;Abca1f/f mice were injected once intravenously with 109 pfu of either AAV-TBG-Luc (Adeno-associated virus expressing luciferase under the control of human thyroxin binding-globulin promoter; control) or AAV-TBG-Cre viruses (to ablate hepatic floxed genes) in 100 µl of sterile PBS. Additionally, mice injected with AAV-TBG-Luc were injected intraperitoneally with 200 µl of corn oil for three alternate days to serve as controls. Mice injected with AAV-TBG-Cre were injected with 0.5 mg of tamoxifen (induces Cre-recombinase expression in the intestine to ablate floxed genes) in 200 µl of corn oil for three alternate days to obtain L,I-Abca1−/− mice. To generate individual liver-specific (L-Abca1−/−) or intestine-specific (I-Abca1−/−) KOs, ERT2-villin-Cre;Abca1f/f mice (34Iqbal J. Parks J.S. Hussain M.M. Lipid absorption defects in intestine-specific microsomal triglyceride transfer protein and ATP-binding cassette transporter A1-deficient mice.J. Biol. Chem. 2013; 288: 30432-30444Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) were either injected once intravenously with 109 pfu of AAV-TBG-Cre virus or intraperitoneally with 0.5 mg of tamoxifen in 200 µl of corn oil for three alternate days, respectively. To generate liver- and intestine-specific KOs for both Mttp and Abca1 genes, we crossed Mttpf/f and ERT2-villin-Cre;Abca1f/f mice to obtain ERT2-villin-Cre;Abca1f/fMttpf/f mice. These mice were injected with AAV-TBG-Cre and tamoxifen as described above. Tissue-specific liver (L-Mttp−/−) or intestine (I-Mttp−/−) KOs were generated by injecting ERT2-villin-Cre;Mttpf/f mice (34Iqbal J. Parks J.S. Hussain M.M. Lipid absorption defects in intestine-specific microsomal triglyceride transfer protein and ATP-binding cassette transporter A1-deficient mice.J. Biol. Chem. 2013; 288: 30432-30444Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) either once intravenously with 109 pfu of AAV-TBG-Cre virus or intraperitoneally with 0.5 mg of tamoxifen in 200 µl of corn oil for three alternate days, respectively. Plasma and tissues were harvested 7 days after the last tamoxifen injection. All experiments were approved by the Institutional Animal Care and Use Committee at SUNY Downstate Medical Center. Kits were used to measure triglycerides (Thermo Fisher Scientific, catalog no. TR22421), phospholipids (Wako Diagnostics, catalog no. 433-36201), and cholesterol (Thermo Fisher Scientific, catalog no. TR13421) in plasma, liver, and intestine according to the manufacturer's instructions. Human apoB and apoAI were quantified by ELISA (35Hussain M.M. Zhao Y. Kancha R.K. Blackhart B.D. Yao Z. Characterization of recombinant human apoB-48-containing lipoproteins in rat hepatoma McA-RH7777 cells transfected with apoB48 cDNA: overexpression of apoB-48 decreases synthesis of endogenous apoB-100.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 485-494Crossref PubMed Scopus (63) Google Scholar, 36Bakillah A. Zhou Z. Luchoomun J. Hussain M.M. Measurement of apolipoprotein B in various cell lines: correlation between intracellular levels and rates of secretion.Lipids. 1997; 32: 1113-1118Crossref PubMed Scopus (38) Google Scholar). Plasma (100 µl) from heterozygous abetalipoproteinemia (normal; n = 4) and HE (n = 5) and HO (n = 6) Tangier subjects, as well as plasma (150 or 100 µl) from different WT and KO mice (n = 3), was used for the quantification of different species of Cer, SM, HxCer, and LactCer using HPLC-MS/MS (1Hammad S.M. Pierce J.S. Soodavar F. Smith K.L. Al Gadban M.M. Rembiesa B. Klein R.L. Hannun Y.A. Bielawski J. Bielawska A. Blood sphingolipidomics in healthy humans: impact of sample collection methodology.J. Lipid Res. 2010; 51: 3074-3087Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 14Iqbal J. Walsh M.T. Hammad S.M. Cuchel M. Tarugi P. Hegele R.A. Davidson N.O. Rader D.J. Klein R.L. Hussain M.M. Microsomal triglycerdie transfer protein transfers and determines plasma concentrations of ceramide and sphingomyelin but not glycosylceramide.J. Biol. Chem. 2015; 290: 25863-25875Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Amounts of different sphingolipid species were combined to obtain total plasma levels. Lipids were extracted from 2 mg of liver or intestinal tissue homogenates from mice and used to quantify intracellular sphingolipids. For detection of MTP or ABCA1 in the liver, liver homogenates were homogenized in Buffer K. They (20 µg of protein) were separated on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane, blocked with 50 mM Tris, pH 7.6, 150 mM NaCl, 0.5% Tween 20 and 5% milk (TBS plus Tween 20), probed with either mouse anti-mouse MTP (BD Transduction LaboratoriesTM, catalog no. 612022; 1:3,000 dilution) or anti-mouse ABCA1 followed by Alexa Fluor® 633-labeled anti-IgG (Life Technologies, Inc.; 1:10,000 dilution). The membranes were stripped and probed with anti-vinculin (Sigma, catalog no. V9131; 1:2,000 dilutions) or anti-GAPDH (Santa Cruz, catalog no. sc-48167; 1:2,000 dilution) antibodies as a control. To detect apoB and apoA1, plasma (0.5 µl) was run on a 5–14% polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was cut in half. One-half was probed with rabbit anti-mouse apoB (Meridian Life Science, catalog no. K23300R; 1:1,000 dilution) and then incubated with Alexa Fluor® 633 goat anti-rabbit IgG (Life Technologies, Inc., catalog no. A-21070; 1:10,000 dilution). The other half was probed with goat anti-apoA1 (Academy Bio-Medical Co., Inc., catalog no. 11S-G2; 1:2,000 dilution) followed by Alexa Fluor® 633 donkey anti-goat IgG (Life Technologies, Inc., catalog no. A-21082; 1:10,000 dilutions). Bands were visualized in a phosphorimager. Huh-7 cells (ATCC) were grown in DMEM supplemented with 10% FBS, L-glutamine, and antibiotics. Cells were reverse-transfected with siRNAs using RNAiMAX (Thermofisher, catalog no. 13778150) according to the manufacturer's instructions. Briefly, siABCA1, scrambled siRNA, and RNAiMAX were diluted individually in serum-reduced Opti-MEM, combined, and incubated at room temperature for 30 min. Next, Huh-7 cells were trypsinized, centrifuged, and plated in a 96-well plate (Cer synthesis) or 6-well plates (Cer and SM synthesis) containing the complexed siRNA and RNAiMAX. Cells were incubated in reduced serum medium for 24 h. Cells were rinsed and incubated for 24 h in 10% FBS DMEM before analysis. Liver homogenates (250 µg of protein) from WT and KO mice were incubated with [3H]dihydrosphingosine (0.05 µCi), dihydrosphingosine (15 µM), FA-free BSA (20 µM), and lignoceroyl-CoA (50 µM) in the presence or absence of Fumonisin B1 (50 µM; an inhibitor of sphingolipids) in a total volume of 200 µl for 30 min at 37°C. The reaction was stopped by adding 200 μl of CHCl3:CH3OH (2:1, v/v). Lipids were extracted and separated on thin-layer silica plates (catalog no. 44931, Analtech, Inc.) using a CHCl3:CH3OH:C6H5CH3:NH4OH:H2O (40:40:20:0.4:1.6, ratios by volume) solvent system (14Iqbal J. Walsh M.T. Hammad S.M. Cuchel M. Tarugi P. Hegele R.A. Davidson N.O. Rader D.J. Klein R.L. Hussain M.M. Microsomal triglycerdie transfer protein transfers and determines plasma concentrations of ceramide and sphingomyelin but not glycosylceramide.J. Biol. Chem. 2015; 290: 25863-25875Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Radioactivities in the dihydrosphingosine and dihydroceramide bands were quantified using a scintillation counter. Liver tissues from WT and KO mice were homogenized in a buffer containing 50 mM Tris-HCl, 1 mM EDTA, 5% sucrose (pH 7.4), and a cocktail of protease inhibitors. The homogenate was centrifuged in a tabletop Centrifuge 5415D (Eppendorf) at 2,320 g for 10 min at 4°C. The supernatant (250 µg of protein) was added to assay buffer containing 50 mM Tris-HCl (pH 7.4), 25 mM KCl, C6-NBD Cer (3.3 µg/ml), and phosphatidylcholine (100 µg/ml) in a total volume of 200 µl reaction and incubated for 2 h at 37°C. The reaction was stopped by adding 200 μl of CHCl3:CH3OH (2:1, v/v). Lipids were extracted (37Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42689) Google Scholar), dried, and resuspended in 100 µl of isopropanol for the separation of sphingolipids on TLC as above. The TLC plates were visualized with the phosphorimager, and bands were quantified. Huh-7 cells or hepatocytes isolated from different mice were incubated with 2 µM C6-NBD Cer in 10% FBS containing DMEM at 37°C for 3 h and washed three times with DMEM plus 0.1% BSA, and the efflux was initiated by the addition of fresh DMEM containing 40 µg/ml either BSA, human serum LDL (catalog no. MBS173145, MyBioSource), human serum ApoA1 (catalog no. MBS173253, MyBioSource), or human serum HDL (catalog no. MBS173147, MyBioSource). In some experiments, efflux was performed in the presence or absence of either 2 µM LXR agonist (T0901317, catalog no. T2320, Sigma-Aldrich) or 500 nM MTP inhibitor (Lomitapide) or 250 µM ABCA1 inhibitor (glyburide, catalog no. 2539, Sigma-Aldrich). For LXR agonist experiments, cells were pretreated with 2 µM LXR agonist for 17 h before the efflux. Culture media were harvested after 6 h of incubation and centrifuged at 580 g for 15 min at 4°C to pellet-detached cells. Lipids in the media were extracted, separated on TLC, and visualized with the phosphorimager as described above. Comparisons between WT and different KO mouse models were made by Student t-test. Comparisons among HE ABL (normal), HE Tangier, and HO Tangier subjects were evaluated using the Student t-test using GraphPad Prism. Significance among different treatments in supplemental tables was determined by one-way ANOVA. MTP plays a critical role in determining plasma concentrations of Cer and SM, but plasma levels of HxCer and LactCer were found not to be affected by MTP deficiency (14Iqbal J. Walsh M.T. Hammad S.M. Cuchel M. Tarugi P. Hegele R.A. Davidson N.O. Rader D.J. Klein R.L. Hussain M.M. Microsomal triglycerdie transfer protein transfers and determines plasma concentrations of ceramide and sphingomyelin but not glycosylceramide.J. Biol. Chem. 2015; 290: 25863-25875Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), even though these sphingolipids are also found to be associated with B-lps (1Hammad S.M. Pierce J.S. Soodavar F. Smith K.L. Al Gadban M.M. Rembiesa B. Klein R.L. Hannun Y.A. Bielawski J. Bielawska A. Blood sphingolipidomics in healthy humans: impact of sample collection methodology.J. Lipid Res. 2010; 51: 3074-3087Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 2Hammad S.M. Blood sphingolipids in homeostasis and pathobiology.Adv. Exp. Med. Biol. 2011; 721: 57-66Crossref PubMed Scopus (39) Google Scholar, 3Hammad S.M. Al Gadban M.M. Semler A.J. Klein R.L. Sphingosine 1-phosphate distribution in human plasma: associations with lipid profiles.J. Lipids. 2012; 2012: 180705Crossref PubMed Google Scholar, 4Wiesner P. Leidl K. Boettcher A. Schmitz G. Liebisch G. Lipid profiling of FPLC-separated lipoprotein fractions by electrospray ionization tandem mass spectrometry.J. Lipid Res. 2009; 50: 574-585Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 5Dawson G. Kruski A.W. Scanu A.M. Distribution of glycosphingolipids in the serum lipoproteins of normal human subjects and patients with hypo- and hyperlipidemias.J. Lipid Res. 1976; 17: 125-131Abstract Full Text PDF PubMed Google Scholar). Here, we hypothesized that HxCer and LactCer arrive to the blood circulation via apoB-independent pathways. We have previously shown that apoB-dependent and -independent pathways contribute to cholesterol absorption (38Iqbal J. Anwar K. Hussain M.M. Multiple, independently regulated pathways of cholesterol transport across the intestinal epithelial cells.J. Biol. Chem. 2003; 278: 31610-31620Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 39Iqbal J. Hussain M.M. Intestinal lipid absorption.Am. J. Physiol. Endocrinol. Metab. 2009; 296: E1183-E1194Crossref PubMed Scopus (529) Google Scholar) and that MTP and ABCA1, respectively, are the major determinants of these pathways (34Iqbal J. Parks J.S. Hussain M.M. Lipid absorption defects in intestine-specific microsomal triglyceride transfer protein and ATP-binding cassette transporter A1-deficient mice.J. Biol. Chem. 2013; 288: 30432-30444Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 39Iqbal J. Hussain M.M. Intestinal lipid absorption.Am. J. Physiol. Endocrinol. Metab. 2009; 296: E1183-E1194Crossref PubMed Scopus (529) Google Scholar). Hence, we investigated whether ABCA1 might contribute to plasma glycosylceramide levels. To investigate whether ABCA1 in humans is important for plasma sphingolipids, we measured these lipids in the plasma of HE and HO Tangier patients that carry codominant mutations in the ABCA1 gene (27Assmann G. Von Eckardstein A. Brewer Jr., H.B. 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