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• W2109376993 abstract "Niemann-Pick C (NPC) disease is a fatal inherited disorder characterized by an accumulation of cholesterol and other lipids in late endosomes/lysosomes. Although this disease is considered to be primarily a neurodegenerative disorder, many NPC patients suffer from liver disease. We have investigated alterations that occur in hepatic lipid homeostasis using primary hepatocytes isolated from NPC1-deficient mice. The cholesterol content of Npc1–/– hepatocytes was 5-fold higher than that of Npc1+/+ hepatocytes; phospholipids and cholesteryl esters also accumulated. In contrast, the triacylglycerol content of Npc1–/– hepatocytes was 50% lower than of Npc1+/+ hepatocytes. We hypothesized that the cholesterol sequestration induced by NPC1 deficiency might inhibit very low density lipoprotein secretion. However, this process was enhanced by NPC1 deficiency and the secreted particles were enriched in cholesteryl esters. We investigated the mechanisms responsible for these changes. The synthesis of phosphatidylcholine, cholesteryl esters, and cholesterol in hepatocytes was increased by NPC1 deficiency and the amount of the mature form of sterol response element-binding protein-1 was also increased. These observations indicate that the enhanced secretion of lipoproteins from NPC1-deficient hepatocytes is due, at least in part, to increased lipid synthesis. Niemann-Pick C (NPC) disease is a fatal inherited disorder characterized by an accumulation of cholesterol and other lipids in late endosomes/lysosomes. Although this disease is considered to be primarily a neurodegenerative disorder, many NPC patients suffer from liver disease. We have investigated alterations that occur in hepatic lipid homeostasis using primary hepatocytes isolated from NPC1-deficient mice. The cholesterol content of Npc1–/– hepatocytes was 5-fold higher than that of Npc1+/+ hepatocytes; phospholipids and cholesteryl esters also accumulated. In contrast, the triacylglycerol content of Npc1–/– hepatocytes was 50% lower than of Npc1+/+ hepatocytes. We hypothesized that the cholesterol sequestration induced by NPC1 deficiency might inhibit very low density lipoprotein secretion. However, this process was enhanced by NPC1 deficiency and the secreted particles were enriched in cholesteryl esters. We investigated the mechanisms responsible for these changes. The synthesis of phosphatidylcholine, cholesteryl esters, and cholesterol in hepatocytes was increased by NPC1 deficiency and the amount of the mature form of sterol response element-binding protein-1 was also increased. These observations indicate that the enhanced secretion of lipoproteins from NPC1-deficient hepatocytes is due, at least in part, to increased lipid synthesis. Niemann-Pick type C (NPC) 2The abbreviations used are: NPC, Niemann-Pick type C; apo, apolipoprotein; CE, cholesteryl esters; CT, CTP:phosphocholine cytidylyltransferase; ER, endoplasmic reticulum; LDL, low density lipoprotein; PC, phosphatidylcholine; SREBP, sterol response element-binding protein; TG, triacylglycerol; VLDL, very low density lipoprotein. disease is an inherited autosomal, recessive disorder that is characterized by an accumulation of cholesterol and other lipids in tissues. Individuals with this disease experience progressive neurodegeneration and premature death, typically during teenage years (1Pentchev P.G. Vanier M.T. Suzuki K. Patterson M.C. Scriver C.R. Baudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 7 Ed. McGraw-Hill Inc., New York1995Google Scholar). The majority (∼95%) of cases of NPC disease result from mutations in the NPC1 gene (2Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. Nagle J. Polymeropoulos M.H. Sturley S.L. Ioannou Y.A. Higgins M.E. et al.Science. 1997; 277: 228-231Crossref PubMed Scopus (1216) Google Scholar), whereas the remaining 5% have mutations in the NPC2 gene (3Naureckiene S. Sleat D.E. Lackland H. Fensom A. Vanier M.T. Wattiaux R. Jadot M. Lobel P. Science. 2000; 290: 2298-2301Crossref PubMed Scopus (701) Google Scholar). The NPC1 gene encodes a transmembrane protein that resides in late endosomes/lysosomes (4Higgins M.E. Davies J.P. Chen F.W. Ioannou Y.A. Mol. Gen. Metab. 1999; 68: 1-13Crossref PubMed Scopus (210) Google Scholar, 5Neufeld E.B. Wastney M. Patel S. Suresh S. Cooney A.M. Dwyer N.K. Roff C.F. Ohno K. Morris J.A. Carstea E.D. Incardona J.P. Strauss J.F. Vanier M.T. Patterson M.C. Brady R.O. Pnetchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1999; 274: 9627-9635Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). The protein contains a sterol-sensing domain, a leucine-zipper motif, and a lysosomal targeting sequence (2Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. Nagle J. Polymeropoulos M.H. Sturley S.L. Ioannou Y.A. Higgins M.E. et al.Science. 1997; 277: 228-231Crossref PubMed Scopus (1216) Google Scholar, 6Watari H. Blanchette-Mackie E.J. Dwyer N.K. Glick J.M. Patel S. Neufeld E.B. Brady R.O. Pnetchev P.G. Strauss J.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 805-810Crossref PubMed Scopus (126) Google Scholar, 7Watari H. Blanchette-Mackie E.J. Dwyer N.K. Watari M. Neufeld E.B. Patel S. Pentchev P.G. Strauss 3rd, J.F. J. Biol. Chem. 1999; 274: 21861-21866Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In NPC1-deficient cells, low density lipoprotein (LDL)-derived cholesterol, as well as gangliosides and other lipids, accumulate in late endosomes/lysosomes. Thus, although the exact function of NPC1 has not yet been established, NPC1 appears to be required for the egress of cholesterol and/or other lipids from the endosomal pathway (8Vanier M.T. Biochim. Biophys. Acta. 1983; 750: 178-183Crossref PubMed Scopus (151) Google Scholar, 9Sokol J. Blanchette-Mackie E.J. Kruth H.S. Dwyer N.K. Amende L.M. Butler J.D. Robinson E. Patel S. Brady R.O. Comly M.E. Vanier M.T. Pentchev P.G. J. Biol. Chem. 1988; 263: 3411-3417Abstract Full Text PDF PubMed Google Scholar, 10Goldin E. Roff C.F. Miller S.P. Rodriguez-Lafrasse C. Vanier M.T. Brady R.O. Pentchev P.G. Biochim. Biophys. Acta. 1992; 1127: 303-311Crossref PubMed Scopus (57) Google Scholar, 11Watanabe Y. Akaboshi S. Ishida G. Takeshima T. Yano T. Taniguchi M. Ohno K. Nakashima K. Brain Dev. 1998; 20: 95-97Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 12Kobayashi T. Beuchat M.H. Lindsay M. Frias S. Palmiter R.D. Sakuraba H. Parton R.G. Gruenberg J. Nat. Cell Biol. 1999; 1: 113-118Crossref PubMed Scopus (249) Google Scholar, 13Zervas M. Somers K.L. Thrall M.A. Walkley S.U. Curr. Biol. 2001; 11: 1283-1287Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). A consequence of the sequestration of cholesterol in NPC1-deficient cells is that normal cholesterol homeostatic responses are impaired. In normal fibroblasts, when the cholesterol content increases, cholesterol biosynthesis is down-regulated and cholesterol esterification is stimulated (14Brown M.S. Goldstein J.L. Science. 1986; 232: 34-47Crossref PubMed Scopus (4362) Google Scholar). In NPC1-deficient fibroblasts, however, cholesterol becomes sequestered in late endosomes/lysosomes and the cholesterol homeostatic machinery in the endoplasmic reticulum (ER) fails to sense the increased level of cellular cholesterol. Consequently, cholesterol synthesis is inappropriately increased and the esterification of LDL-derived cholesterol is markedly decreased (15Liscum L. Faust J.R. J. Biol. Chem. 1987; 262: 17002-17008Abstract Full Text PDF PubMed Google Scholar, 16Liscum L. Ruggiero R.M. Faust J.R. J. Cell Biol. 1989; 108: 1625-1636Crossref PubMed Scopus (241) Google Scholar). In addition to the severe, progressive neurodegeneration in individuals with NPC disease, hepatomegaly and neonatal cholestasis occur in many NPC patients. A significant number of children with NPC disease die of liver failure within their first six months (17Kelly D.A. Portmann B. Mowat A.P. Sherlock S. Lake B.D. J. Pediatr. 1993; 123: 242-247Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 18Yerushalmi B. Sokol R.J. Narkewicz M.R. Smith D. Ashmead J.W. Wenger D.A. J. Pediatr. Gastroenterol. Nutr. 2002; 35: 44-50Crossref PubMed Scopus (82) Google Scholar, 19Vanier M.T. Millat G. Clin. Genet. 2003; 64: 269-281Crossref PubMed Scopus (492) Google Scholar, 20Beltroy E.P. Richardson J.A. Horton J.D. Turley S.D. Dietschy J.M. Hepatology. 2005; 42: 886-893Crossref PubMed Scopus (104) Google Scholar). Because the majority of cholesterol-containing LDLs are cleared from the circulation by the liver via receptor-mediated endocytosis (21Pittman R.C. Steinberg D. J. Lipid Res. 1984; 25: 1577-1585Abstract Full Text PDF PubMed Google Scholar, 22Xie C. Turley S.D. Dietschy J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11992-11997Crossref PubMed Scopus (86) Google Scholar), NPC deficiency causes an accumulation of cholesterol in the livers of humans and mice to a greater extent than in any other tissue (22Xie C. Turley S.D. Dietschy J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11992-11997Crossref PubMed Scopus (86) Google Scholar, 23Osono Y. Woollett L.A. Herz J. Dietschy J.M. J. Clin. Investig. 1995; 95: 1124-1132Crossref PubMed Scopus (164) Google Scholar). Mice lacking functional NPC1 represent an excellent model for studying NPC disease because they exhibit liver dysfunction, including hepatomegaly and elevation of plasma alkaline phosphatase and aminotransferase levels, similar to those seen in infants with NPC disease (20Beltroy E.P. Richardson J.A. Horton J.D. Turley S.D. Dietschy J.M. Hepatology. 2005; 42: 886-893Crossref PubMed Scopus (104) Google Scholar). In light of these liver problems, we have investigated alterations in lipid metabolism that occur in primary hepatocytes isolated from NPC1-deficient mice. Cholesterol levels in Npc1–/– hepatocytes are markedly (∼5-fold) higher than in Npc1+/+ hepatocytes and the rates of cholesterol esterification and the synthesis of cholesterol and phosphatidylcholine (PC) are increased. Moreover, NPC1 deficiency increases the amount of the mature form of sterol response element-binding protein (SREBP-1) and enhances the secretion of very low density lipoproteins (VLDLs) by hepatocytes. Materials—Hanks' solution, collagenase, penicillin, streptomycin, and fetal bovine serum were obtained from Invitrogen. Phenylmethylsulfonyl fluoride, protein A-Sepharose CL-4B, tridecanoin standard for gas chromatography, phospholipase C from Clostridium welchii, and insulin were purchased from Sigma, as was the inhibitor of acyl-CoA:cholesterol acyltransferase, Sandoz 58-035. Complete protease inhibitor tablets were from Roche. [3H]Glycerol (3 Ci/mmol), [14C]acetate (57 mCi/mmol), [2-14C]mevalonic acid lactone, [9,10-3H]oleic acid, and [methyl-3H]choline (75.7 Ci/mmol) were from Amersham Biosciences. All chemicals used for polyacrylamide gel electrophoresis were from Bio-Rad. Goat anti-human apolipoprotein (apo) B polyclonal antibodies were purchased from Chemicon (San Diego, CA). Mouse anti-goat IgG linked to horseradish peroxidase was obtained from Pierce. The rabbit anti-rat phosphatidylethanolamine N-methyltransferase-2 polyclonal antibodies were generated in our laboratory (24Cui Z. Vance J.E. Chen M.H. Voelker D.R. Vance D.E. J. Biol. Chem. 1993; 268: 16655-16663Abstract Full Text PDF PubMed Google Scholar). The rabbit anti-human CTP:phosphocholine cytidylyltransferase (CT) polyclonal antibodies (25Lykidis A. Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) were generously provided by Dr. S. Jackowski (St. Jude Children's Research Institute, Memphis, TN). The rabbit anti-canine calnexin antibodies were purchased from StressGen (Vancouver, British Columbia, Canada). The rabbit antibodies directed against human SREBP-1 and SREBP-2 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Mouse anti-rabbit IgG linked to horseradish peroxidase was from Pierce. The chemiluminescent reagent used for immunoblotting was from Amersham Biosciences. Cholesterol, cholesteryl esters (CE), triacylglycerols (TG), PC, and sphingomyelin used as thin layer chromatography standards were either purchased from Avanti Polar Lipids (Alabaster, AL) or isolated in our laboratory from rat livers. BIOCOAT collagen-coated cell culture dishes (60 mm) and thin layer chromatography plates (Silica Gel G, 0.25 mm thickness) were from VWR (Mississauga, ON, Canada), as was the Infinity Cholesterol reagent and the Triacylglycerol reagent used for detection of lipids in the column eluate from fast protein liquid chromatography. All other chemicals and reagents were from Fisher Scientific or Sigma. Culture of Primary Hepatocytes from Npc1-deficient Mice—Male mice (5 weeks old) were from a breeding colony of Balb/cNctr-NpcN/+ mice established at the University of Alberta from original breeding pairs obtained from The Jackson Laboratories (Bar Harbor, ME). The mice were maintained under temperature-controlled conditions with a 12-h light, 12-h dark cycle, and were supplied with a 9% fat diet (number 5001 from Purina LabDiet, Richmond, IN) and water ad libitum. Henceforth, mice homozygous or heterozygous for the Npc1 mutation are referred to as Npc1–/– and Npc1+/–, respectively, whereas wild-type mice are termed Npc1+/+. Because Npc1–/– mice do not produce offspring, Npc1+/– mice were used for breeding. The Npc1 genotype was determined from tail clippings by PCR analysis of genomic DNA using primers described previously (26Karten B. Vance D.E. Campenot R.B. Vance J.E. J. Neurochem. 2002; 83: 1154-1163Crossref PubMed Scopus (130) Google Scholar). For isolation of hepatocytes, mice were anesthetized by intraperitoneal injection of Somnotol (22 μl/50 g body weight). A mid-line incision was made, and Hanks' EGTA solution containing 1 mg/ml insulin was perfused through the portal vein until the liver was clear of blood. The upper and lower vena cava were tied and the perfusion was continued with Hanks' collagenase solution (100 units/ml) containing 1 mg/ml insulin until the liver became soft (∼3 min). The liver was removed, cut into pieces, transferred to Hanks' collagenase solution, and mixed until all clumps of tissue dispersed. The hepatocytes were washed three times in Dulbecco's modified Eagle's medium, then suspended in medium containing 10% fetal bovine serum and plated on collagen-coated dishes (1 × 106 cells/ml). Cell viability (typically >90% for both Npc1+/+ and Npc1–/– hepatocytes) was estimated by trypan blue exclusion. After hepatocytes had attached to the dish (3–4 h), the medium was removed and cells were incubated in medium lacking serum. Filipin Staining of Npc1+/+ and Npc1–/– Hepatocytes—Hepatocytes were cultured on collagen-coated coverslips for 16 h then fixed for 20 min at room temperature in 4% paraformaldehyde. Phosphate-buffered saline containing 10% goat serum and 50 μg/ml filipin was added for 2 h at room temperature. Pictures were taken using a Leica DM IRE2 fluorescence microscope (Leica, ON, Canada) with an excitation wavelength of 351 nm. Mass of Lipids in Hepatocytes and Culture Medium—Hepatocytes from one 60-mm culture dish were scraped into water (1 ml) and lipids were extracted (27Folch J. Lees M. Sloane-Stanley G.H. J. Biol. Chem. 1959; 226: 495-509Google Scholar). The culture medium (2 ml) was also collected, cell debris was removed by centrifugation, and lipids were extracted (27Folch J. Lees M. Sloane-Stanley G.H. J. Biol. Chem. 1959; 226: 495-509Google Scholar). The amounts of TG, CE, and unesterified cholesterol were determined in hepatocyte lysates and in culture medium. After digestion of phospholipids with phospholipase C, tridecanoin (20 ng) was added as an internal standard. The mass of TG, cholesterol, and CE was determined by gas-liquid chromatography (26Karten B. Vance D.E. Campenot R.B. Vance J.E. J. Neurochem. 2002; 83: 1154-1163Crossref PubMed Scopus (130) Google Scholar). For measurement of the mass of phospholipids, lipids were extracted from an aliquot of liver homogenate (2 mg of protein) or culture medium (2 ml) (27Folch J. Lees M. Sloane-Stanley G.H. J. Biol. Chem. 1959; 226: 495-509Google Scholar) and separated by thin layer chromatography in the solvent system chloroform/methanol/acetic acid/formic acid/water (70:30:12:4:1, v/v). Bands corresponding to authentic PC and sphingomyelin were visualized by exposure of the plate to iodine and spots were scraped from the plate. Amounts of PC and sphingomyelin were quantified by phosphorus analysis (28Zhou X. Arthur G. J. Lipid Res. 1992; 33: 1233-1236Abstract Full Text PDF PubMed Google Scholar). Isolation of VLDLs from Culture Medium and Measurement of Lipid Content—Npc1+/+ and Npc1–/– hepatocytes were incubated for 16 h in Dulbecco's modified Eagle's medium in the absence of serum. Culture medium (1.3 ml) was collected and mixed with 0.7 g of KBr. The sample was placed in a 5.0-ml Quick-seal tube and overlaid with 3.5 ml of 0.9% NaCl. The samples were centrifuged at 416,000 × g for 1 h in a Beckmann VTi 90.0 rotor (29Kulinski A. Vance D.E. Vance J.E. J. Biol. Chem. 2004; 279: 23916-23924Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Fractions containing high density lipoproteins, LDLs, and VLDLs were sequentially collected from the bottom of the tube. The density of each fraction was determined as the weight of 1.0 ml. Medium from 5 dishes of cells was combined. The mass of TG and CE was determined by gas-liquid chromatography. ApoB Content of Hepatocytes, Culture Medium, and Plasma—Hepatocytes from one 60-mm culture dish were scraped into 750 μl of phosphate-buffered saline, then 250 μl of buffer containing Tris-HCl (0.66 m, pH 7.4), NaCl (0.75 m), EDTA (25 mm), phenylmethylsulfonyl fluoride (5 mm), and Triton X-100 (5%, v/v) was added. Cellular extracts were centrifuged in a microcentrifuge at 14,000 × g for 10 min, after which proteins in the supernatant were immunoprecipitated by incubation overnight at 4 °C with anti-human apoB antibodies (7.5 μl). Protein A-Sepharose (45 mg) was added and the sample was mixed end-over-end for 2 h at 4 °C. The apoB-protein A complexes were pelleted by centrifugation for 2 min at 14,000 × g in a microcentrifuge. For immunoprecipitation of secreted apoB, culture medium was centrifuged for 2 min at 1,000 × g to remove cell debris and anti-apoB antibodies were added to the supernatant. Equal amounts of protein in the immunoprecipitate were separated by electrophoresis on 5% polyacrylamide gels in the presence of 0.4% SDS, then transferred to polyvinylidene difluoride membranes. Proteins were immunoblotted with goat anti-human apoB antibodies and subsequently with anti-goat IgG linked to horseradish peroxidase. Immunoreactive proteins were detected by chemiluminescence. The amount of apoB was quantified by densitometric scanning of the blots and calculated as intensity of the band per mg of cell protein. The apoB content of plasma was compared in Npc1+/+ and Npc1–/– mice that had been fasted for 16 h. Plasma (100 μl) was collected and lipoproteins were separated on the basis of density by ultracentrifugation on a KBr gradient (29Kulinski A. Vance D.E. Vance J.E. J. Biol. Chem. 2004; 279: 23916-23924Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Ten fractions with densities ranging from 1.21 (fraction 10) to 1.006 g/ml (fraction 1) were collected. ApoB was immunoprecipitated from equal volumes of each fraction as described above, and proteins were separated by electrophoresis on 7.5% polyacrylamide gels containing 0.1% SDS. ApoB100 and apoB48 were analyzed by immunoblotting. Separation of Lipoproteins on the Basis of Size from Hepatocyte Culture Medium—Hepatocytes were isolated from livers of male 5-week-old Npc1+/+ and Npc1–/– mice and incubated in Dulbecco's modified Eagle's medium for 16 h. Culture medium from four, 100-mm culture dishes was combined and concentrated to a volume of 200 μl with an Amicon Ultra Concentrator (100,000 molecular weight cut-off from Millipore). Lipoproteins were separated on the basis of size by fast protein liquid chromatography on a Superose 6 column (30Jacobs R.L. Devlin C. Tabas I. Vance D.E. J. Biol. Chem. 2004; 279: 47402-47410Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The amount of total cholesterol (i.e. unesterified cholesterol plus esterified cholesterol) and TG in the column eluate was measured with Infinity Cholesterol reagent or Triacylglycerol reagent, respectively. The elution time of the lipid reflects the size of the lipoprotein particles. Metabolic Labeling of PC, Cholesterol, CE, and TG—Hepatocytes were incubated for up to 3 h with Dulbecco's modified Eagle's medium containing 5 μCi/ml [methyl-3H]choline. Alternatively, hepatocytes were incubated with [14C]acetate (20 μCi/ml), [3H]oleate (50 μCi/ml), [14C]mevalonic acid lactone (5 μCi/ml), or [3H]glycerol (5 μCi/ml), as indicated. Cells were washed twice with Dulbecco's modified Eagle's medium and lipids were extracted. PC was isolated by thin layer chromatography in the developing solvent chloroform/methanol/acetic acid/formic acid/water (70:30:12:4:1, v/v). The band corresponding to authentic PC was visualized by exposure of the plate to iodine, then scraped from the plate and radioactivity was measured. Cholesterol, CE, and TG were isolated by thin layer chromatography with heptane/isopropyl ether/acetic acid (60:40:4, v/v) as developing solvent. Esterification of Cholesterol—Npc1+/+ and Npc1–/– hepatocytes were incubated for 16 h in Dulbecco's modified Eagle's medium in the absence of serum. The cells were then incubated with [3H]oleic acid (50 μCi/ml) bound to 163 mg/ml fatty acid-free albumin for up to 4 h. The cells were washed twice with phosphate-buffered saline. Lipids were extracted (27Folch J. Lees M. Sloane-Stanley G.H. J. Biol. Chem. 1959; 226: 495-509Google Scholar) then separated by thin layer chromatography in heptane/isopropyl ether/acetic acid (60:40:4, v/v) as developing solvent. Lipids were visualized by exposure of the plate to iodine vapor and the band corresponding to CE was scraped from the plate for measurement of radioactivity. In control experiments, cholesterol esterification was assessed in human skin fibroblasts using the same protocol. Npc1+/+ fibroblasts were purchased from the American Type Culture Collection (Manassas, VA) and Npc1–/– fibroblasts were from the Human Genetic Mutant Cell Repository (Camden, NJ). The fibroblasts were grown to near confluence in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, then cholesterol esterification was assessed as described above. In some experiments, cholesterol esterification was assessed in Npc1+/+ and Npc1–/– hepatocytes by incubation of cells with [3H]oleate for 2 h in the presence or absence of Sandoz 58-035 (2 μg/ml), an inhibitor of acyl-CoA:cholesterol acyltransferase. Immunoblotting of CTP:phosphocholine Cytidylyltransferase, Phosphatidylethanolamine N-Methyltransferase, Calnexin, SREBP-1, and SREBP-2—Homogenates of livers from Npc1+/+ and Npc1–/– mice were centrifuged at 10,000 × g for 10 min to remove cell debris and mitochondria. The supernatant was centrifuged at 90,000 × g for 30 min to pellet membranes. Membrane proteins were separated by electrophoresis on 10% polyacrylamide gels containing 0.1% SDS, then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 10% skimmed milk, then incubated overnight with anti-CT antibodies (1:1,000 dilution) that recognize both CTα and CTβ; in hepatocytes the α isoform predominates. The membranes were incubated with anti-rabbit IgG linked to horseradish peroxidase (dilution 1:2,500). Immunoreactive proteins were visualized by enhanced chemiluminescence. Essentially the same procedure was used for immunoblotting of calnexin, SREBP-1, and SREBP-2, except that anti-calnexin antibodies were used at a dilution of 1:5,000, and SREBP-1 and SREBP-2 antibodies were used at a dilution of 1:250. For immunoblotting of phosphatidylethanolamine methyltransferase, proteins in liver homogenates were separated by electrophoresis on 12% polyacrylamide gels containing 0.1% SDS, then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 10% skimmed milk, then incubated overnight with anti-phosphatidylethanolamine methyltransferase antibodies (1:1,000 dilution) (24Cui Z. Vance J.E. Chen M.H. Voelker D.R. Vance D.E. J. Biol. Chem. 1993; 268: 16655-16663Abstract Full Text PDF PubMed Google Scholar), followed by incubation with anti-rabbit IgG linked to horseradish peroxidase (dilution 1:2,500). Immunoreactive bands were visualized by enhanced chemiluminescence. Other Methods—The protein content of samples was determined using the BCA protein assay (Pierce) with bovine serum albumin as standard. Statistical significance of difference was determined by the Student's t test and differences were considered significant at p < 0.05. Accumulation of Lipids in NPC1-deficient Hepatocytes—The distribution of unesterified cholesterol in primary hepatocytes isolated from 5-week-old Npc1+/+ and Npc1–/– mice was assessed by staining the cells with filipin (26Karten B. Vance D.E. Campenot R.B. Vance J.E. J. Neurochem. 2002; 83: 1154-1163Crossref PubMed Scopus (130) Google Scholar, 31Karten B. Vance D.E. Campenot R.B. Vance J.E. J. Biol. Chem. 2003; 278: 4168-4175Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), an agent that detects un-esterified cholesterol but not CE. Fluorescence microscopy of filipin-treated Npc1+/+ hepatocytes revealed that un-esterified cholesterol was localized mainly to the cell surface (Fig. 1A), whereas Npc1–/– hepatocytes showed an intense, punctate intracellular staining pattern, indicative of redistribution of unesterifed cholesterol (Fig. 1B). These filipin staining patterns are reminiscent of those seen in other types of Npc1+/+ and Npc1–/– cells such as fibroblasts (32Cadigan K.M. Spillane D.M. Chang T.Y. J. Cell Biol. 1990; 110: 295-308Crossref PubMed Scopus (91) Google Scholar, 33Roff C.F. Goldin E. Comly M.E. Blanchette-Mackie J. Cooney A. Brady R.O. Pentchev P.G. Am. J. Med. Genet. 1992; 42: 593-598Crossref PubMed Scopus (40) Google Scholar), neurons (26Karten B. Vance D.E. Campenot R.B. Vance J.E. J. Neurochem. 2002; 83: 1154-1163Crossref PubMed Scopus (130) Google Scholar), and glial cells (34Karten B. Hayashi H. Francis G.A. Campenot R.B. Vance D.E. Vance J.E. Biochem. J. 2005; 387: 779-788Crossref PubMed Scopus (35) Google Scholar). To establish the extent of unesterified cholesterol accumulation in NPC1-deficient hepatocytes, we measured the mass of cholesterol by gas-liquid chromatography. In Npc1–/– hepatocytes, the cholesterol content was ∼5-fold higher than in Npc1+/+ hepatocytes (Fig. 1C). Thus, in hepatocytes, as in other types of cells, lack of functional NPC1 causes a pronounced re-distribution and accumulation of unesterified cholesterol. It is noteworthy that in mouse hepatocytes NPC1 deficiency causes a much greater accumulation of cholesterol than in other types of cells such as fibroblasts (25–50% increase) (35Frolov A. Zielinski S.E. Crowley J.R. Dudley-Rucker N. Schaffer J.E. Ory D.S. J. Biol. Chem. 2003; 278: 25517-25525Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), glial cells (∼20% increase) (34Karten B. Hayashi H. Francis G.A. Campenot R.B. Vance D.E. Vance J.E. Biochem. J. 2005; 387: 779-788Crossref PubMed Scopus (35) Google Scholar), and in cell bodies of neurons (∼40% increase) (26Karten B. Vance D.E. Campenot R.B. Vance J.E. J. Neurochem. 2002; 83: 1154-1163Crossref PubMed Scopus (130) Google Scholar). The amounts of several other lipids, PC, sphingomyelin, CE, and TG, in Npc1+/+ and Npc1–/– hepatocytes were also compared. The amounts of PC (Fig. 2A), sphingomyelin (Fig. 2B), and CE (Fig. 2C) were significantly higher (by 48, 100, and 76%, respectively) in Npc1–/– hepatocytes than in Npc1+/+ hepatocytes. In contrast, the mass of cellular TG in Npc1–/– hepatocytes was 54% less (p < 0.001) than in wild-type hepatocytes (Fig. 2D). The cholesterol and CE content of livers of 5-week-old Npc1+/+ and Npc1–/– mice was also compared. The amount of unesterified cholesterol in NPC1-deficient livers was 5-fold higher than in wild-type livers (142.0 ± 24.9 versus 28.7 ± 3.9 μg/mg of protein and the amount of hepatic CE was not significantly changed by NPC1 deficiency (2.15 ± 1.41 versus 1.49 ± 0.33 μg/mg protein). NPC1 Deficiency in Hepatocytes Increases the Secretion of Unesterified Cholesterol, CE, and Phospholipids, but Not TG—An important function of hepatocytes is to provide lipids for lipoprotein secretion. The majority of lipids secreted by cultured primary hepatocytes are associated with VLDLs although small amounts of cholesterol, CE, PC, and sphingomyelin are also released into the medium as high density lipoproteins. PC, cholesterol, and sphingomyelin reside primarily on the surface monolayer of lipoprotein particles, whereas the neutral lipids TG and CE are in the core. The mass of cholesterol (Fig. 3A), PC (Fig. 3B), and sphingomyelin (Fig. 3C) secreted into the culture medium of NPC1-deficient hepatocytes was increased by NPC1 deficiency (by 117, 48, and 74%, respectively). In addition, the amount of CE in the culture medium of Npc1–/– hepatocytes was ∼50% (p < 0.008) higher than in the medium of Npc1+/+ hepatocytes (Fig. 3D). However, the mass of TG secreted was unchanged by NPC1 deficiency (Fig. 3E). These observations suggest that NPC1 deficiency in hepatocytes increases VLDL secretion. Furthermore, the data indicate that the composition of the neutral lipid core of the VLDLs might have been modifie" @default.
• W2109376993 created "2016-06-24" @default.
• W2109376993 creator A5019669813 @default.
• W2109376993 creator A5091291303 @default.
• W2109376993 date "2007-01-01" @default.
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