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Chylomicrons produced by the human gut contain apolipoprotein (apo) B48, whereas very-low-density lipoproteins made by the liver contain apo B100. To study how these molecules function during lipid absorption, we examined the process as it occurs in apobec-1 knockout mice (able to produce only apo B100; KO) and in wild-type mice (of which the normally functioning intestine makes apo B48, WT). Using the lymph fistula model, we studied the process of lipid absorption when animals were intraduodenally infused with a lipid emulsion (4 or 6 micromol/h of triolein). KO mice transported triacylglycerol (TG) as efficiently as WT mice when infused with the lower lipid dose; when infused with 6 micromol/h of triolein, however, KO mice transported significantly less TG to lymph than WT mice, leading to the accumulation of mucosal TG. Interestingly, the size of lipoprotein particles from both KO and WT mice were enlarged to chylomicron-size particles during absorption of the higher dose. These increased-size particles produced by KO mice were not associated with increased apo AIV secretion. However, we found that the gut of the KO mice secreted fewer apo B molecules to lymph (compared with WT), during both fasting and lipid infusion, leading us to conclude that the KO gut produced fewer numbers of TG-rich lipoproteins (including chylomicron) than the wild-type animals. The reduced apo B secretion in KO mice was not related to reduced microsomal triglyceride transfer protein lipid transfer activity. We propose that apo B48 is the preferred protein for the gut to coat chylomicrons to ensure efficient chylomicron formation and lipid absorption.
Rat hepatic Golgi apparatus-rich fractions were utilized in an in vitro phosphorylation system containing [gamma-32P]ATP to investigate the phosphorylation of apolipoproteins (apo) B48 and B100. Our results demonstrate that the Golgi apparatus contains a kinase(s) that phosphorylates both apoB48 and apoB100 as well as 290- and 460-kDa proteins recognized by antibody to apoB. We refer to the latter proteins as apoB57 and apoB90, respectively. Phosphorylations in the presence of Triton X-100, which increases the permeability of the membranes, or alamethicin, an ionophore that facilitates transmembrane diffusion of ATP, indicate that the active site of the kinase is on the luminal side of the membranes. However, studies with EDTA and EGTA, which are inhibitory to the kinase, suggest binding sites for Mg2+ and perhaps Ca2+ on the cytosolic membrane face. Phosphorylation of apoB was not stimulated by cAMP nor inhibited by protein kinase inhibitor peptide (5-24), indicating that cAMP dependent protein kinase was not involved in the phosphorylation process. Sodium carbonate treatment of the phosphorylated fraction, which permits separation of membrane and luminal contents, revealed that phosphorylated apoB90 and apoB57 are associated primarily with the membrane, whereas phosphorylated apoB48 is found in luminal contents as well as with the membranes. Phosphorylated apoB100 was found primarily with the membrane fraction. No evidence was found for phosphorylation of apoB in rough endoplasmic reticulum fractions. These studies demonstrate the importance of the Golgi apparatus as a subcellular site for the phosphorylation of apoB and suggest that apoB phosphorylation may be important in the assembly and secretion of apoB-containing lipoproteins.
Transgenic mice that express human apolipoprotein (apo)B have been developed by microinjecting fertilized mouse oocytes with an 80 kb genomic DNA fragment that encompasses the entire human APOB gene. In the transgenic mice expressing the largest amounts of human apoB, the concentration of human apoB100 in the plasma is nearly as high as the levels observed in normolipidemic humans (50 mg/dl). Virtually all of the human apoB100 in the transgenic plasma is located in the LDL fraction, resulting in substantially increased levels of LDL cholesterol. These human apoB-transgenic mice should be useful animal models for understanding various aspects of lipoprotein metabolism and for further delineating the role of LDL in atherogenesis.
To investigate the assembly pathway for hepatic very low density lipoproteins (VLDL), nascent lipoproteins were recovered from a purified, intact rough endoplasmic reticulum (ER) fraction isolated from rat liver. Two fractions were recovered by ultracentrifugation. Particles isolated at d 1.006 g/ml were triglyceride-rich particles containing apolipoprotein (apo)B-100 or apoB-48, and apoE with very small amounts of apoA-I. Compared with VLDL recovered from the Golgi apparatus, the particles from the rough ER had less triglyceride, but more cholesteryl ester and phospholipid. The second class of particles isolated between d 1.006 and 1.210 g/ml were phospholipid-rich and contained apoB-48, apoE, and apoA-I. ApoB-100 was a minor component. Radioisotope incorporation studies utilizing [3H]leucine revealed differential rates of labeling of the apoproteins in these two lipoprotein fractions. ApoB-100 and apoE followed similar patterns in both fractions with peak incorporation occurring within 15 min of isotope injection. Incorporation of [3H]leucine into apoB-48 in the dense fraction peaked within 15 min of isotope administration, but peak incorporation in the d 1.006 g/ml fraction did not occur until approximately 30 min after injection. We propose that the two lipoprotein fractions recovered from the rough ER are intermediates in the assembly of VLDL by the liver. Comparison of the composition of these two particles with that of Golgi VLDL supports the sequential assembly of VLDL by the liver. Furthermore, we propose that the initial steps in the assembly of apoB-100- and apoB-48-containing lipoproteins are different with nascent apoB-100-containing particles being formed through the cotranslational association of this apoprotein with lipid while nascent apoB-48-containing VLDL are formed in the rough ER through a two-step process.
The synthesis of apoB-100 and apoB-48 by rat liver was investigated by studying the apoB complement of very low density lipoproteins (VLDL) from hepatic perfusates and Golgi fractions. The relative amounts of apoB-100 and apoB-48 in perfusate and Golgi VLDL as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis were similar to those in serum VLDL. To investigate the relative rates of synthesis of the VLDL B proteins, rats were injected intraportally with tritiated amino acid, and hepatic Golgi and serum VLDL were isolated from 7.5 to 120 min later. In hepatic Golgi VLDL, apoB-100 and apoE were maximally labeled at 15 min after the tritiated amino acid pulse. In contrast, VLDL apoB-48 attained maximum radioactivity at 30 min after isotope injection. In serum VLDL, apoB-100 and apoE were maximally labeled at 30 min post-isotope injection, while activity in apoB-48 peaked at 60 min. The data suggest that the synthesis of the B proteins and incorporation into rat liver nascent VLDL are independently regulated. The differential labeling patterns of the VLDL B proteins may be explained by an intracellular pool of apoB-48 that is larger than that of apoB-100. An alternative explanation of the results is that apoB-100 is a precursor to apoB-48.
A primed-constant infusion of deuterated leucine was used in humans to determine the maximal level of enrichment at plateau of apolipoprotein (apo)B-48 and apoB-100 which are synthesized in the intestine and liver, respectively, and to compare the kinetics of these two proteins under identical conditions. Eight normal subjects (four post-menopausal females and four males) over the age of 40 were studied in the constantly fed state over a 20-h period by providing small hourly feedings of identical composition. [5,5,5-2H3]Leucine (10 mumol/kg body weight followed by 10 mumol/kg body weight per hour) was infused over 15 h intravenously. The enrichment of deuterated leucine in apoB-48 and apoB-100 triglyceride-rich lipoproteins isolated by ultracentrifugation (d less than 1.006 g/ml) was determined during the entire infusion period. The plateau level of enrichment in triglyceride-rich lipoprotein apoB-48 was 3.96 +/- 1.41 tracer/tracee ratio (%) which was 39.7% of the plasma leucine enrichment level. The plateau level of enrichment in triglyceride-rich lipoprotein apoB-100 was 7.23 +/- 1.17 tracer/tracee ratio (%) which was 72.5% of the plasma leucine enrichment level. Mean fractional secretion rates of triglyceride-rich lipoprotein apoB-48 and apoB-100 were 4.39 +/- 2.00 and 5.39 +/- 1.98 pools per day, respectively, with estimated residence times of 5.47 and 4.45 hours, respectively. The data indicate that in the fed state there is about a twofold difference in the plateau enrichment of an intestinally derived protein, as compared to one of hepatic origin, most likely attributable to differences in the enrichment of the intracellular leucine in the two organs.(ABSTRACT TRUNCATED AT 250 WORDS)