Spontaneously diabetic Ins2+/Akita:apoE-deficient mice exhibit exaggerated hypercholesterolemia and atherosclerosis
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Type 1 diabetes (T1D) increases the risk of adverse coronary events. Among risk factors, dyslipidemia due to altered hepatic lipoprotein metabolism plays a central role in diabetic atherosclerosis. Nevertheless, the likely alterations in plasma lipid/lipoprotein profile remain unclear, especially in the context of spontaneously developed T1D and atherosclerosis. To address this question, we generated Ins2(+/Akita):apoE(-/-) mouse by cross-breeding Ins2(+/Akita) mouse (which has Ins2 gene mutation, causing pancreatic β-cell apoptosis and insulin deficiency) with apoE(-/-) mouse. Ins2(+/Akita):apoE(-/-) mice developed T1D spontaneously at 4-5 wk of age. At 25 wk of age and while on a standard chow diet, diabetic Ins2(+/Akita):apoE(-/-) mice exhibited an approximately threefold increase in atherosclerotic plaque in association with an approximatelty twofold increase in plasma non-HDL cholesterol, predominantly in the LDL fraction, compared with nondiabetic controls. To determine factors contributing to the exaggerated hypercholesterolemia, we assessed hepatic VLDL secretion and triglyceride content, expression of hepatic lipoprotein receptors, and plasma apolipoprotein composition. Diabetic Ins2(+/Akita):apoE(-/-) mice exhibited diminished VLDL secretion by ~50%, which was accompanied by blunted Akt phosphorylation in response to insulin infusion and decreased triglyceride content in the liver. Although the expression of hepatic LDL receptor was not affected, there was a significant reduction in the expression of lipolysis-stimulated lipoprotein receptor (LSR) by ~28%. Moreover, there was a marked decrease in plasma apoB-100 with a significant increase in apoB-48 and apoC-III levels. In conclusion, exaggerated hypercholesterolemia and atherosclerosis in spontaneously diabetic Ins2(+/Akita):apoE(-/-) mice may be attributable to impaired lipoprotein clearance in the setting of diminished expression of LSR and altered apolipoprotein composition of lipoproteins.Keywords:
Apolipoprotein E
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In the present study it was investigated whether apolipoprotein (apoE) can inhibit the lipoprotein lipase (LPL)-mediated hydrolysis of very-low-density-lipoprotein (VLDL) triacylglycerols (TAGs). Previous studies have suggested such an inhibitory role for apoE by using as a substrate for LPL either plasma VLDL or artificial TAG emulsions. To mimic the in vivo situation more fully, we decided to investigate the effect of apoE on the LPL-mediated TAG hydrolysis by using VLDL from apoE-deficient mice that had been enriched with increasing amounts of apoE. Furthermore, since plasma VLDL isolated from apoE-deficient mice was relatively poor in TAGs and strongly enriched in cholesterol as compared with VLDL from wild-type mice, we used nascent VLDL obtained by liver perfusions. Nascent VLDL (d < 1.006) isolated from the perfusate of the apoE-deficient mouse liver was rich in TAGs. Addition of increasing amounts of apoE to apoE-deficient nascent VLDL effectively decreased TAG lipolysis as compared with that of apoE-deficient nascent VLDL without the addition of apoE (63.1±6.3 and 20.8±1.8% of the control value at 2.7 μg and 29.6 μg of apoE/mg of TAG added respectively). Since, in vivo, LPL is attached to heparan sulphate proteoglycans (HSPG) at the endothelial matrix, we also performed lipolysis assays with LPL bound to HSPG in order to preserve the interaction of the lipoprotein particle with the HSPG-LPL complex. In this lipolysis system a concentration-dependent decrease in the TAG lipolysis was also observed with increasing amounts of apoE on nascent VLDL, although to a lesser extent than with LPL in solution (72.3±3.6% and 56.6±1.7% of control value at 2.7μg and 29.6 μg of apoE/mg TAGs added respectively). In conclusion, the enrichment of the VLDL particle with apoE decreases its suitability as a substrate for LPL in a dose-dependent manner.
Apolipoprotein E
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Intermediate-density lipoprotein
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Internalization of apoE-containing very low density protein (VLDL) by hepatocytes in vivo and in vitro leads to apoE recycling and resecretion. Because of the role of apoE in VLDL metabolism, apoE recycling may influence lipoprotein assembly or remnant uptake. However, apoE is also a HDL protein, and apoE recycling may be related to reverse cholesterol transport. To investigate apoE recycling, apoE−/− mouse hepatocytes were incubated (pulsed) with wild-type mouse lipoproteins, and cells and media were collected at chase periods up to 24 h. When cells were pulsed with VLDL, apoE was resecreted within 30 min. Although the mass of apoE in the media decreased with time, it could be detected up to 24 h after the pulse. Intact intracellular apoE was also detectable 24 h after the pulse. ApoE was also resecreted when cells were pulsed with HDL. When apoA-I was included in the chase media after a pulse with VLDL, apoE resecretion increased 4-fold. Furthermore, human apoE was resecreted from wild-type mouse hepatocytes after a pulse with human VLDL. Finally, apoE was resecreted from mouse peritoneal macrophages after pulsing with VLDL. We conclude that 1) HDL apoE recycles in a quantitatively comparable fashion to VLDL apoE; 2) apoE recycling can be modulated by extracellular apoA-I but is not affected by endogenous apoE; and 3) recycling occurs in macrophages as well as in hepatocytes, suggesting that the process is not cell-specific.
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We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [3H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 ± 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in thed < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum. We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [3H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 ± 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in thed < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum. apolipoprotein endoplasmic reticulum very low density lipoprotein(s) high density lipoprotein(s) polyacrylamide gel electrophoresis Immunoelectron microscopy studies have shown that apolipoprotein (apo)1 B is present in the rough endoplasmic reticulum (ER), but very low density lipoprotein (VLDL)-sized particles are not (1.Alexander C.A. Hamilton R.L. Havel R.J. J. Cell Biol. 1976; 69: 241-263Crossref PubMed Scopus (253) Google Scholar). VLDL particles with immunoreactive apoB first appeared in the smooth termini of the rough ER; the smooth ER contained VLDL-sized particles without immunoreactive apoB (1.Alexander C.A. Hamilton R.L. Havel R.J. J. Cell Biol. 1976; 69: 241-263Crossref PubMed Scopus (253) Google Scholar). Based on these results, a two-step model for the assembly of VLDL was proposed. Dynamic evidence for this model was obtained by pulse-chase studies of apoB-100 and apoB-48 (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar, 3.Swift L.L. J. Lipid Res. 1995; 36: 395-406Abstract Full Text PDF PubMed Google Scholar). The first step occurs during the translation of apoB and gives rise to a partially lipidated form of apoB (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar, 4.Borén J. Graham L. Wettesten M. Scott J. White A. Olofsson S.-O. J. Biol. Chem. 1992; 267: 9858-9867Abstract Full Text PDF PubMed Google Scholar). In the case of apoB-100, this partially lipidated particle appeared to be loosely associated with the ER membrane (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In the case of apoB-48, a particle resembling high density lipoprotein (HDL) has been identified. The secretion of this dense, apoB-48-containing, HDL-like lipoprotein varied inversely with that of VLDL (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). Therefore, we hypothesized that this particle is a precursor of apoB-48 VLDL (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). A second VLDL precursor was identified as an apoB-free "lipid droplet" in the smooth ER (1.Alexander C.A. Hamilton R.L. Havel R.J. J. Cell Biol. 1976; 69: 241-263Crossref PubMed Scopus (253) Google Scholar, 6.Hamilton R.L. Wong J.S. Cham C.M. Nielsen L.B. Young S.G. J. Lipid Res. 1998; 39: 1543-1557Abstract Full Text Full Text PDF PubMed Google Scholar). The assembly of both precursors is dependent on the microsomal triglyceride transfer protein (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 7.Gordon D.A. Jamil H. Gregg R.E. Olofsson S.-O. Borén J. J. Biol. Chem. 1996; 271: 33047-33053Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar,8.Raabe M. Véniant M.M. Sullivan M.A. Zlot C.H. Björkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (362) Google Scholar). The mechanism for the second step, fusion of the two precursors (1.Alexander C.A. Hamilton R.L. Havel R.J. J. Cell Biol. 1976; 69: 241-263Crossref PubMed Scopus (253) Google Scholar), is less well understood. We have demonstrated that brefeldin A inhibits the major lipidation of apoB (9.Rustaeus S. Lindberg K. Borén J. Olofsson S.-O. J. Biol. Chem. 1995; 270: 28879-28886Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). However the exact localization of the brefeldin A-sensitive mechanism in the assembly pathway remains to be elucidated. Cotranslational or early post-translational degradation of apoB is important in regulating the amount of apoB that passes through the first step (10.Zhou M. Wu X. Huang L.S. Ginsberg H.N. J. Biol. Chem. 1995; 270: 25220-25224Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 11.Fisher E.A. Zhou M. Mitchell D.M. Wu X. Omura S. Wang H. Goldberg A.L. Ginsberg H.N. J. Biol. Chem. 1997; 272: 20427-20434Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). This degradation involves ubiquitination and proteasomes (11.Fisher E.A. Zhou M. Mitchell D.M. Wu X. Omura S. Wang H. Goldberg A.L. Ginsberg H.N. J. Biol. Chem. 1997; 272: 20427-20434Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Recent results indicate that apoB is completely translocated to the lumen of the ER (12.Shelness G.S. Morris-Rogers K.C. Ingram M.F. J. Biol. Chem. 1994; 269: 9310-9318Abstract Full Text PDF PubMed Google Scholar), suggesting that the early post-translational degradation follows the pathway described for misfolded proteins (i.e. the protein is retracted through the translocation channel) (13.Cresswell P. Hughes E.A. Curr. Biol. 1997; 7: 552-555Abstract Full Text Full Text PDF PubMed Google Scholar). However, there is strong evidence that the degradation involves nascent as well as full-length apoB chains (14.Zhou M. Fisher E.A. Ginsberg H.N. J. Biol. Chem. 1998; 273: 24649-24653Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 15.Liao W. Yeung S.-C.J. Chan L. J. Biol. Chem. 1998; 273: 27225-27230Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), suggesting that this proteasomal degradation may also involve other pathways (for reviews, see Refs. 16.Ginsberg H.N. Clin. Exp. Pharmacol. Physiol. 1997; 24: A29-A32Crossref PubMed Scopus (49) Google Scholar and 17.Olofsson S.-O. Asp L. Borén J. Curr. Opin. Lipidol. 1999; 10: 341-346Crossref PubMed Scopus (188) Google Scholar). Studies of VLDL assembly have been hampered by the fact that much of the apoB present in the cell remains associated with the microsomal membrane after carbonate extraction of the luminal proteins and therefore cannot be analyzed. Recently, we developed a procedure that extracts virtually all of the apoB-100 from the microsomal membranes without releasing integral membrane proteins (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In this study, we used this new extraction procedure in a series of experiments to analyze the assembly of apoB-48 VLDL. Eagle's minimum essential medium, nonessential amino acids, glutamine, penicillin, and streptomycin were obtained from ICN Biomedicals (Costa Mesa, CA). Fetal calf serum was from Biochrom KG (Berlin, Germany) and brefeldin A from Epicenter Technologies (Madison, WI). Methionine, fatty acid-free bovine serum albumin, sodium pyruvate, disodium carbonate, sodium hydrogen carbonate, phenylmethylsulfonyl fluoride, pepstatin A, and leupeptin were from Sigma. Rabbit immunoglobulin was from Dako (Glostrup, Denmark), and rabbit anti-rat transferrin IgG was from Organon Teknika (West Chester, PA). Trasylol (aprotinin) was from Bayer (Leverkusen, Germany). Immunoprecipitin and Eagle's minimum essential medium without methionine were from Life Technologies, Inc. N-Acetyl-Leu-Leu-norleucinal as well as enzymatic assays for the determination of phospholipids or triglycerides were from Boehringer Mannheim. Amplify, [35S]methionine/cysteine mix, Rainbow protein molecular weight markers, and the ECL Western blotting analysis system were from Amersham Pharmacia Biotech, and Ready-Safe was from Beckman (Fullerton, CA). All chemicals used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline phosphatase-conjugated goat anti-rabbit and rabbit anti-mouse were from Bio-Rad (Hercules, CA). Blue-stabilized substrate for alkaline phosphatase and trypsin (sequencing grade) were from Promega (Milwaukee, WI). Cyanogen bromide-activated Sepharose 4B was from Amersham Pharmacia Biotech, and α-cyano-4-OH cinnamic acid was from Aldrich (Milwaukee, WI). Antibodies to chaperones (binding protein, protein disulfide isomerase, glucose regulatory protein 94, and calreticulin) were purchased from Affinity BioReagents (Golden, CO). McA-RH7777 cells were cultured as described previously (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar) in Eagle's minimum essential medium containing 20% fetal calf serum, 1.6 mm glutamine, 8.0 mmNaHCO3, 1.6 mm sodium pyruvate, 140 mg/ml streptomycin, 140 IU/ml penicillin, and 60 mg/ml nonessential amino acids in 5% CO2 at 37 °C. The cultures were split twice a week and fed daily. The cells were pulse labeled and chased as described (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). Cells and the microsomal fraction were isolated as described (18.Boström K. Borén J. Wettesten M. Sjöberg A. Bondjers G. Wiklund O. Carlsson P. Olofsson S.-O. J. Biol. Chem. 1988; 263: 4434-4442Abstract Full Text PDF PubMed Google Scholar). The luminal content of the vesicles was separated from the vesicle membranes by the sodium carbonate method (19.Fujiki Y. Hubbard A.L. Fowler S. Lazarow P. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1385) Google Scholar), as modified (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). The following protease inhibitors were used: 0.1 mmleupeptin, 1 mm phenylmethylsulfonyl fluoride, 1 mm pepstatin A, 5 mm N-acetyl-Leu-Leu-norleucinal, and aprotinin (100 kallekrein-inhibitory units/ml). In some experiments, the luminal content of the microsomal vesicles was extracted with the deoxycholate/carbonate procedure described recently by Rustaeuset al. (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The total microsomal fraction was obtained (18.Boström K. Borén J. Wettesten M. Sjöberg A. Bondjers G. Wiklund O. Carlsson P. Olofsson S.-O. J. Biol. Chem. 1988; 263: 4434-4442Abstract Full Text PDF PubMed Google Scholar) and fractionated on a sucrose gradient. A 3.8-ml linear sucrose gradient (32.5–40%, w/v) was layered on a cushion of 0.5 ml of 65% sucrose (w/v), and the sample was layered on top of the gradient. All solutions contained 3 mm imidazole, pH 7.4, with the same protease inhibitors as used for metabolic labeling. The gradients were centrifuged in a Beckman Vti-65.2 vertical rotor at 50,000 rpm for 3 h at 12 °C. The gradient was unloaded from the bottom into 22 fractions. NADPH cytochrome c reductase and galactosyl transferase were used as marker enzymes for the ER and the Golgi apparatus, respectively (20.Borén J. Wettesten M. Sjöberg A. Thorlin T. Bondjers G. Wiklund O. Olofsson S.-O. J. Biol. Chem. 1990; 265: 10556-10564Abstract Full Text PDF PubMed Google Scholar). Gradient fractions were also assayed by Western blot for calnexin, a marker for the rough ER. Lipoproteins in the microsomal lumen or in the medium were separated by sucrose gradient ultracentrifugation (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). The gradient was formed by layering, from the bottom of the tube, 2 ml of 49% sucrose, 2 ml of 25% sucrose, 5 ml of the sample in 12.5% sucrose (the sucrose was in phosphate-buffered saline), and 3 ml of phosphate-buffered saline. All solutions contained 0.1 mm leupeptin, 1 mm phenylmethylsulfonyl fluoride, 1 mm pepstatin A, 5 mm N-acetyl-Leu-Leu-norleucinal, aprotinin (100 kallekrein-inhibitory units/ml), and 0.5 mm EDTA. The gradients were centrifuged in a Beckman SW40 rotor at 35,000 rpm for 65 h at 12 °C and unloaded from the bottom of the tube into 12–13 fractions. ApoB was immunoprecipitated from the cells, medium, and sucrose gradient fractions as described (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar, 21.Wettesten M. Boström K. Bondjers G. Jarfeldt M. Norfeldt P.-I. Carrella M. Wiklund O. Borén J. Olofsson S.-O. Eur. J. Biochem. 1985; 149: 461-466Crossref PubMed Scopus (32) Google Scholar). Immunoaffinity chromatography of apoB-containing fractions extracted from the microsomes or present in the culture medium was carried out as described (18.Boström K. Borén J. Wettesten M. Sjöberg A. Bondjers G. Wiklund O. Carlsson P. Olofsson S.-O. J. Biol. Chem. 1988; 263: 4434-4442Abstract Full Text PDF PubMed Google Scholar). SDS-PAGE, autoradiography, and determination of the radioactivity in proteins separated in the gels were performed as detailed elsewhere (21.Wettesten M. Boström K. Bondjers G. Jarfeldt M. Norfeldt P.-I. Carrella M. Wiklund O. Borén J. Olofsson S.-O. Eur. J. Biochem. 1985; 149: 461-466Crossref PubMed Scopus (32) Google Scholar). Lipids from McA-RH7777 cells were extracted as described by Olegård and Svennerholm (22.Olegård R. Svennerholm L. Acta Paediatr. Scand. 1970; 59: 637-647Crossref PubMed Scopus (108) Google Scholar) with slight modifications (23.Andersson M. Wettesten M. Borén J. Magnusson A. Sjöberg A. Rustaeus S. Olofsson S.-O. J. Lipid Res. 1994; 35: 535-545Abstract Full Text PDF PubMed Google Scholar). Phosphatidylcholine and triglycerides were separated by thin layer chromatography with chloroform:methanol:water (65:25:4 v/v) followed by petroleumether:diethylether:acetic acid (80:20:1 v/v). The spots were visualized by iodine, scraped off, and extracted with 1 ml of chloroform:methanol 1:2 (phosphatidylcholine) or chloroform (triglycerides). The extracted lipids were dried under nitrogen in a conical tube, solubilized in 20 μl of ethanol, and analyzed by enzymatic assays. As standards, pure phosphatidylcholine and triglycerides were chromatographed and processed in parallel with the sample. The recovery of triglycerides and phosphatidylcholine during the chromatography and extraction steps was 94 ± 16% (mean ± S.D.; n = 5) and 65 ± 4% (n = 5). The recovery of triglycerides was also tested with radioactive tracer added to the cell homogenate; this experiment showed a recovery of 94 ± 8% (n = 4). The intra-assay variation was 4.5% for triglycerides and 8.1% for phosphatidylcholine. Phosphatidylcholine and triglycerides were radiolabeled by incubating the cells for various periods with [3H]glycerol (0.6 μCi/ml of culture medium). Cells were extracted, phosphatidylcholine and triglycerides were separated as described above, and specific radioactivity was determined (dpm/mg). In some experiments, apoB-containing lipoproteins were isolated by immunoaffinity chromatography from the luminal content or the medium. During the extraction of the lipids from these fractions, unlabeled phosphatidylcholine and triglycerides were added as carriers. The lipids were separated as described above, and the spots corresponding to triglycerides and phosphatidylcholine were scraped into scintillation vials; 1 ml of cyclohexane was added, and the radioactivity was determined in the presence of Ready-Safe scintillation mixture. Proteins associated with microsomal lipoproteins were isolated and identified as follows. Rat liver microsomes were isolated (23.Andersson M. Wettesten M. Borén J. Magnusson A. Sjöberg A. Rustaeus S. Olofsson S.-O. J. Lipid Res. 1994; 35: 535-545Abstract Full Text PDF PubMed Google Scholar), and the luminal content was extracted with sodium carbonate (19.Fujiki Y. Hubbard A.L. Fowler S. Lazarow P. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1385) Google Scholar). The extract (6 ml) was overlayered with 29 ml of phosphate-buffered saline (8 mm disodium hydrogen phosphate, 1.5 mmpotassium dihydrogen phosphate, 137 mm sodium chloride, and 2.7 mm potassium chloride, pH 7.4, d = 1.006 g/ml). After centrifugation for 22 h at 40,000 rpm in a Beckman Ti-60 rotor at 4 °C, the gradients were fractionated from the top. The upper one-third of the tube (d < 1.006 g/ml) was collected. Pooled fractions of this supernatant (corresponding to three to five rat livers) were loaded onto a Mono Q column equilibrated with 50 mm Tris-HCl, pH 7.8, with 300 mm sucrose, 1 mm EDTA, 2 mmdeoxycholate, 0.5% Triton X-100, 6 m urea, and 40 mm sodium carbonate. The column was eluted with a linear gradient of sodium chloride (0–250 mm) at a flow rate of 0.5 ml/min. Fractions (0.5 ml) were collected, and the proteins in each fraction were separated by SDS-PAGE on 10% gels. Gels were stained with silver. Fractions containing the same protein patterns were combined, concentrated, and subjected to SDS-PAGE on 3–15% gradient gels. The gels were stained with Coomassie Brilliant Blue, and the bands were cut out, destained with 50 μl of a mixture of 50% ammonium bicarbonate (25 mm) and 50% acetonitrile, dried, and digested for 15 min with 0.1–0.2 mg of trypsin in 20 μl of 50% ammonium bicarbonate (25 mm) and 50% acetonitrile. Ammonium bicarbonate (25 mm, pH 8) was added to cover the gels, and incubation was continued for 12 h at 37 °C. Fragments were extracted with 10–50 μl of a mixture of 75% acetonitrile and 5% trifluoroacetic acid (in water). Mass spectra were obtained on a TofSpec-E time-of-flight mass spectrometer (Micromass; Manchester, UK) equipped with a time-lagged focusing unit; TOF2UI version 3.4 was used for data collection and OPUS version 3.4 for data analysis. α-Cyano-4-OH cinnamic acid (10 mg/ml in water/acetonitrile, 50/50, v/v) was used as matrix without further purification. The α-cyano-4-OH cinnamic acid solution (0.5 ml) was mixed with the gel extract (0.5 ml) on the target and allowed to dry at room temperature. Spectra were collected in reflectron mode at an accelerating voltage of 20 kV with a 600-ns delayed extraction and a pulse of approximately 2.4 kV. Approximately 200 nitrogen laser pulses (3 ns, 337 nm) were carried out on each sample. For external calibration, a mixture of ACTH and angiotensin II was used (protonated 2465.2 and 1046.5, respectively). The peptide mass fingerprinting software program MS-Fit was run over the Internet. Monoisotopic masses were used for the searches; mass tolerance was ± 200 ppm. To determine the effects of deoxycholate/carbonate extraction (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) on integral microsomal proteins, we first generated a monoclonal antibody against riboforin. BALB/c mice were immunized with solubilized microsomal membrane proteins from rat liver (23.Andersson M. Wettesten M. Borén J. Magnusson A. Sjöberg A. Rustaeus S. Olofsson S.-O. J. Lipid Res. 1994; 35: 535-545Abstract Full Text PDF PubMed Google Scholar). Positive hybridomas were identified by enzyme-linked immunosorbent assay with the antigen (solubilized rat liver microsomes) and analyzed by Western blot of solubilized rat liver microsomes. One hybridoma reacted with a 60-kDa protein and was recloned to monoclonality. The immunoglobulins were isolated from the hybridoma culture medium and coupled to cyanogen bromide-activated Sepharose 4B as recommended by the manufacturer (Amersham Pharmacia Biotech) and used for immunoadsorption experiments (23.Andersson M. Wettesten M. Borén J. Magnusson A. Sjöberg A. Rustaeus S. Olofsson S.-O. J. Lipid Res. 1994; 35: 535-545Abstract Full Text PDF PubMed Google Scholar). Using this immunoadsorbent, we recovered the 60-kDa protein that reacted with the monoclonal antibody. This protein was cut out of the Coomassie-stained gel, digested with trypsin, and analyzed by mass spectrometry as described above. A data search identified the protein as rat riboforin. In contrast to ordinary sodium carbonate extraction, the deoxycholate/carbonate procedure (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) extracted virtually all of the apoB-48 (93 ± 1%; n = 5) from the microsomes. As judged from immunoblot studies of calnexin (see Ref. 5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and riboforin (Fig. 1 A) deoxycholate/carbonate extraction did not release integral membrane proteins. Upon gradient ultracentrifugation, the major amount of apoB-48 extracted by the deoxycholate/carbonate method migrated in the HDL density range (Fig. 1 B). Using a modified gradient, we compared the densities of secreted apoB-48 and deoxycholate/carbonate-extracted apoB-48. The major amount of the secreted apoB-48 was present in the VLDL and the HDL density regions, as described previously (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). ApoB-48 that banded in the HDL density region (apoB-48 HDL) migrated in the same position as the apoB-48 extracted from the microsomes (Fig. 1 C); we will refer to this form of apoB-48 as intracellular apoB-48 HDL. Thus, intracellular apoB-48 HDL and secreted apoB-48 HDL have very similar buoyant densities, and each migrated in the gradient in the expected position for a lipoprotein (in comparison with a nonlipidated protein of similar molecular weight) (Fig. 1 C, III). Thus, the membrane-associated apoB-48, like apoB-100 (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), was extracted from the microsomes as a tentative lipoprotein. To determine if intracellular apoB (both B-100 and B-48)-containing HDL is associated with lipids and contains a lipid core, we began by estimating the incubation time needed to obtain steady-state labeling of the phosphatidylcholine and triglyceride pools of the cell. The cells were incubated with [3H]glycerol (0.6 μCi/ml of culture medium) for 0, 1, 2, 5, 8, and 22 h. After each incubation, phosphatidylcholine and triglycerides were isolated, and the specific radioactivity (dpm/mg) was determined. Triglycerides reached a plateau after 8 h; phosphatidylcholine plateaued between 1 and 8 h, after which the specific radioactivity decreased (data not shown). We therefore incubated the cells for 8 h with [3H]glycerol (0.6 μCi/ml culture of medium). The specific radioactivities of the total phosphatidylcholine (4,025 dpm/μg of lipid) and triglyceride (2,044 dpm/μg of lipid) pools of the cell were determined. The apoB-containing lipoproteins in the HDL density region of the deoxycholate/carbonate extract of the microsomes were isolated by immunoaffinity chromatography, and the radioactivity in phosphatidylcholine and triglycerides was determined. Assuming that the specific radioactivity of the glycerolipids in this apoB fraction was the same as that of the total cell, we estimated the weight ratio between triglycerides and phosphatidylcholine in intracellular apoB HDL to be 0.85 ± 0.15 (n = 3; molar ratio, 0.70 ± 0.09), indicating less triglyceride than phospholipid. Thus, intracellular apoB HDL has an immature lipid core. The labeled cells were also chased for 2 h, and apoB HDL in the medium was isolated by gradient ultracentrifugation followed by immunoaffinity chromatography. Analyzed as described above, the weight ratio between triglycerides and phosphatidylcholine was 0.27 ± 0.10 (n = 3; molar ratio 0.22 ± 0.08), indicating that this particle has a lipid core. Next, we performed pulse-chase experiments to follow the turnover of intracellular apoB-48 HDL and correlated the findings with VLDL assembly and the appearance of apoB-48 HDL in the medium. The cells were pulse labeled with [35S]methionine/cysteine for 10 min and chased for 0–120 min. Radioactive apoB-48 was first seen in the microsomal intracellular apoB-48 HDL (Fig.2). Not until maximal apoB-48 radioactivity was reached in this fraction did any significant amount of apoB-48 radioactivity appear in the VLDL fraction. In fact, the decrease in the apoB-48 radioactivity which followed this maximum accounted for the increased radioactivity in apoB-48 VLDL and in secreted apoB-48 HDL. To determine the length of apoB required for VLDL assembly, we performed pulse-chase studies of nascent apoB chains. To obtain a continuous series of apoB polypeptides of different lengths which could be tested in the assembly process, we truncated apoB with cycloheximide, detached the nascent polypeptides from the ribosomes with puromycin, and chased them through the secretory pathway of the cell into the medium (4.Borén J. Graham L. Wettesten M. Scott J. White A. Olofsson S.-O. J. Biol. Chem. 1992; 267: 9858-9867Abstract Full Text PDF PubMed Google Scholar). The cells were pulse labeled for 10 min and chased for 0–30 min. After each chase period, the cells were treated with cycloheximide and puromycin and then chased for another 180 min in the presence of cycloheximide and puromycin to allow the nascent chains to form lipoproteins and be secreted into the medium. The medium (containing full-length apoB-100/48 as well as the nascent apoB polypeptides that were released into the secretory pathway and secreted during the 180-min chase) was subjected to gradient ultracentrifugation. ApoB was recovered from each fraction by immunoprecipitation and analyzed
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Mipomersen is a 20mer antisense oligonucleotide (ASO) that inhibits apolipoprotein B (apoB) synthesis; its low-density lipoprotein (LDL)-lowering effects should therefore result from reduced secretion of very-low-density lipoprotein (VLDL). We enrolled 17 healthy volunteers who received placebo injections weekly for 3 weeks followed by mipomersen weekly for 7 to 9 weeks. Stable isotopes were used after each treatment to determine fractional catabolic rates and production rates of apoB in VLDL, IDL (intermediate-density lipoprotein), and LDL, and of triglycerides in VLDL. Mipomersen significantly reduced apoB in VLDL, IDL, and LDL, which was associated with increases in fractional catabolic rates of VLDL and LDL apoB and reductions in production rates of IDL and LDL apoB. Unexpectedly, the production rates of VLDL apoB and VLDL triglycerides were unaffected. Small interfering RNA-mediated knockdown of apoB expression in human liver cells demonstrated preservation of apoB secretion across a range of apoB synthesis. Titrated ASO knockdown of apoB mRNA in chow-fed mice preserved both apoB and triglyceride secretion. In contrast, titrated ASO knockdown of apoB mRNA in high-fat-fed mice resulted in stepwise reductions in both apoB and triglyceride secretion. Mipomersen lowered all apoB lipoproteins without reducing the production rate of either VLDL apoB or triglyceride. Our human data are consistent with long-standing models of posttranscriptional and posttranslational regulation of apoB secretion and are supported by in vitro and in vivo experiments. Targeting apoB synthesis may lower levels of apoB lipoproteins without necessarily reducing VLDL secretion, thereby lowering the risk of steatosis associated with this therapeutic strategy.
Catabolism
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Intermediate-density lipoprotein
Apolipoprotein C2
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Studies of truncated apoB peptides in human subjects with familial hypobetalipoproteinemia, as well as of puromycin-generated spectra of nascent apoB peptides in rat and hamster liver, suggest that a minimum size is required for N-terminal fragments of apoB to be efficiently assembled into full-sized VLDL. We report here results of experiments undertaken to examine this phenomenon in greater detail by expressing individual carboxyl-truncated human apoB constructs in McArdle cells. Thus, apoB-29, -32, -37, -42, -47, -53, -70 and full length apoB-100 were transiently expressed in rat McA-RH7777 hepatoma cells, or human apoB-31 and apoB-53 were stably expressed in the same cells, and the secreted VLDL particles were characterized by kinetic gradient ultracentrifugal flotation. Calibration with rat plasma VLDL subfractions showed that about 90 and 50%, respectively, of lipoprotein particles containing endogenous rat B-100 and B-48 floated between fractions 2–8 of the 11-fraction gradient. This corresponds to the normal VLDL diameter range of about 47 to 28 nm, with the remaining half of rat B-48 recovered as HDL particles in the 1.1 g/ml range. In contrast, regardless of their size, only 2–5% of any of the truncated human apoB peptides expressed in these cells was recovered in the VLDL region of the gradient. The remaining 95+% of the lipoproteins were found as high density particles; as previously found in other systems the densities of the latter were inversely related to their peptide chain-length. Furthermore, transiently expressed full-length human apoB-100 was inefficiently secreted as VLDL by these cells, with the remainder appearing as LDL-sized particles. Thus, although we showed that McA-RH7777 cells secreted endogenous rat apoB as normal-sized VLDL, we found them unsuitable for our original purpose of using human apoB fragments to further define effects of apoB size on VLDL assembly. These cells appeared unable to efficiently use any size of human apoB for that process. Pulse-labeled untransfected McA-RH7777 cells chased in the presence of puromycin did, however, show a sharp decline in VLDL assembly efficiency for endogenous nascent rat apoB peptides shorter than B-48, similar to that originally found in normal rat liver. —Xiao, Q., J. Elovson, and V. N. Schumaker. Rat McA-RH7777 cells efficiently assemble rat apolipoprotein B-48 or larger fragments into VLDL but not human apolipoprotein B of any size.
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Apolipoprotein B-100 is a constant component of very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL) in mammalian blood plasma. We have found that each of these classes of lipoproteins includes particles that contain apolipoprotein E (B,E particles) as well as particles that lack this protein (B particles). These two species can be separated by immunosorption on columns of anti-apolipoprotein E bound to Sepharose. We have injected radioiodinated VLDL, IDL, and LDL intravenously into recipient rabbits and have determined the concentration of radioiodine in apolipoprotein B-100 in B,E and B particles in whole-blood plasma obtained at intervals for 24 hr. We have developed a multicompartmental model that is consistent with this new information and with current concepts of lipoprotein metabolism. The model indicates that all apolipoprotein B-100 enters the blood as VLDL, of which about 90% is in B,E particles. Most VLDL B,E particles are removed rapidly from the blood, and only a small fraction is converted to IDL and eventually to LDL (overall conversion is approximately 2%). By contrast, a much smaller fraction of VLDL B particles is removed directly, and approximately 27% is converted to LDL. In addition, some B,E particles are converted to B particles as VLDL are converted to LDL, so that most LDL particles lack apolipoprotein E. Fractional rates of irreversible removal of B,E and B particles in IDL and LDL are similar. Our results indicate that the presence of apolipoprotein E is a major determinant of the metabolic fate of VLDL particles and support the hypothesis that polyvalent binding of particles containing several molecules of apolipoprotein E promotes receptor-dependent endocytosis of hepatogenous lipoproteins and limits their conversion to lipoproteins of higher density.
Intermediate-density lipoprotein
Apolipoprotein C2
Apolipoprotein E
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Apolipoprotein E (apoE) is hypothesized to mediate lipoprotein clearance by binding to two receptors: (i) the low density lipoprotein receptor (LDLR) and (ii) a chylomicron remnant receptor. To test this hypothesis, we have compared plasma lipoproteins in mice that are homozygous for targeted disruptions of the genes for apoE [apoE(-/-)], the LDLR [LDLR(-/-)], and both molecules [poE(-/-); LDLR(-/-)]. On a normal chow diet, apoE(-/-) mice had higher mean plasma cholesterol levels than LDLR(-/-) mice (579 vs. 268 mg/dl). Cholesterol levels in the apoE(-/-); LDLR(-/-) mice were not significantly different from those in the apoE(-/-) mice. LDLR(-/-) mice had a relatively isolated elevation in plasma LDL, whereas apoE(-/-) mice had a marked increase in larger lipoproteins corresponding to very low density lipoproteins and chylomicron remnants. The lipoprotein pattern in apoE(-/-); LDLR(-/-) mice resembled that of apoE(-/-) mice. The LDLR(-/-) mice had a marked elevation in apoB-100 and a modest increase in apoB-48. In contrast, the apoE(-/-) mice had a marked elevation in apoB-48 but not in apoB-100. The LDLR(-/-); apoE(-/-) double homozygotes had marked elevations of both apolipoproteins. The observation that apoB-48 increases more dramatically with apoE deficiency than with LDLR deficiency supports the notion that apoE binds to a second receptor in addition to the LDLR. This conclusion is also supported by the observation that superimposition of a LDLR deficiency onto an apoE deficiency [apoE(-/-); LDLR(-/-) double homozygotes] does not increase hypercholesterolemia beyond the level observed with apoE deficiency alone.
Apolipoprotein E
Chylomicron
Low-density lipoprotein
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Brefeldin A (BFA) added to primary cultures of rat hepatocytes, at a concentration of 0.2 μg/ml, prevented the assembly of newly synthesized apolipoprotein B (apoB) into mature, secretory VLDL but did not prevent the secretion of apoB as denser particles (HDL apoB), or of albumin. The unassembled apoB remained associated with the membranes of the cellular microsomal fraction. There was no effect of BFA on the removal of apoB from the lumen of these vesicles. VLDL apoB formed only a minor component of the total apoB in the microsomal lumen. Higher (5 μg/ml) concentrations of BFA were required to prevent the secretion of HDL apoB and albumin. Under these conditions apoB accumulated in the microsomal lumen, as well as in the membranes of these vesicles. Again, apoB VLDL formed only a minor proportion of the total lumenal apoB. ApoB-48 VLDL and apoB-100 VLDL assembly could be restored by removing BFA from the medium. This reactivation of VLDL assembly was accompanied by an increased removal of apoB from the microsomal membranes, but there was no detectable increase in the small quantity of VLDL apoB that was recovered from the microsomal lumen. In the absence of BFA, during pulse-chase experiments the pattern of change in the specific radioactivity of microsomal membrane apoB was similar to that of the secreted VLDL apoB whereas that of the lumenal apoB resembled that of the secreted HDL apoB.The results suggest that membrane-associated apoB is the main direct precursor of secreted VLDL apoB in primary cultures of rat hepatocytes and that VLDL assembly does not involve primarily microsomal lumenal apoB as an intermediate.
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