Faculty Bibliography

The effect of saturated and unsaturated lipids on the composition of mesenteric lymph triglyceride-rich lipoproteins was studied in rats. A short-term steady-state infusion model was developed in mesenteric lymph fistula rats. Micellar solutions of linoleate, oleate, or palmitate were infused intraduodenally. Steady-state conditions of lymph flow, triglyceride, and apoA-I and apoB secretion rates were achieved in hours 3-5 after the start of the infusion. During this steady-state period, triglyceride-rich lipoproteins were prepared and characterized. With lipid infusion there were the expected increases in secretion rates of triglyceride, apoB, and apoA-I both in whole lymph and in the d less than 1.006 g/ml lipoproteins. Compositional analysis of d less than 1.006 g/ml lipoproteins revealed no difference in the ratios of phospholipid or apoA-I (surface) to triglyceride (core) constituents between saturated and unsaturated lipids, suggesting a similar particle size. This was directly verified by agarose gel filtration and electron microscopy carried out at 27 degrees C, which showed no difference in particle size between linoleate and palmitate chylomicrons. When these lipoproteins were prepared at 4 degrees C, palmitate lipoproteins exhibited dramatically changed gel filtration elution profiles, suggesting a shift to smaller or at least distorted particles and questioning earlier results suggesting a smaller size for saturated fat d less than 1.006 g/ml lipoproteins. Despite the similarity of size between saturated and unsaturated chylomicrons, the apoB content of unsaturated linoleate chylomicrons was significantly lower than that of palmitate chylomicrons. This difference was present whether chylomicrons were prepared by centrifugation or by gel filtration. The clearance of palmitate chylomicrons from the circulation of recipient rats was slightly more rapid than that of linoleate chylomicrons. The mechanism for this apparently selective increase in the apoB content of saturated fat chylomicrons is unknown but the present studies suggest that these changes may be of physiologic significance, perhaps relating to the potential atherogenicity of saturated lipids

Techniques were developed to provide direct quantitation of apolipoprotein A-I (apoA-I) synthesis rates in rat small intestine. Following intralumenal administration of a pulse of [3H]leucine, newly synthesized enterocyte apoA-I was quantitated by specific immunoprecipitation and compared to [3H]leucine incorporation into total trichloroacetic acid-precipitable protein. ApoA-I synthesis rates (% total protein) were found to be significantly higher in jejunal enterocytes (1.84 +/- 0.20) compared to ileal enterocytes (0.91 +/- 0.25) from the same, fasting, animals, P less than 0.01. It was found that rats consuming regular (4.5% w/w fat) rodent chow had apoA-I synthesis rates, 30 to 240 min after receiving an intraduodenal bolus of 100 mg of triglyceride, that were indistinguishable from control animals receiving either saline or an isocaloric, but fat-free, enteral preparation. By contrast, animals consuming a fat-free chow for 8 days prior to study had a small but significant response to acute reintroduction of dietary triglyceride. Four hours after 100 mg of triglyceride was administered, jejunal apoA-I synthesis (% total protein) was 1.84 +/- 0.1 compared to 1.37 +/- 0.04 for animals exposed to an isocaloric, fat-free enteral preparation, P less than 0.01. External bile diversion for 48 hr, which effectively removed all lumenal sources of lipid, reduced apoA-I synthesis in jejunal enterocytes but produced no more depression than that found in sham-operated controls infused for 48 hr with dextrosesaline or control animals fasted for 30 hr. By contrast, apoA-I synthesis in ileal enterocytes was reduced significantly by external bile diversion (0.59 +/- 0.20) in comparison to sham-operated controls (1.19 +/- 0.32) P less than 0.01. Continuous infusion of 10 mM Na taurocholate for 48 hr or 10 mM Na taurocholate for 44 hr and 80 mg of micellar lipid for 4 hr produced results similar to those obtained by bile diversion alone (0.56 +/- 0.2 and 0.61 +/- 0.25, respectively) suggesting that bile salt deficiency alone was not responsible for the observed depression in ileal apoA-I synthesis. These results suggest that, under conditions of physiological dietary triglyceride intake, apoA-I synthesis in jejunal enterocytes is not actuely regulated by changes in triglyceride flux. After prolonged dietary triglyceride withdrawal, the reintroduction of fat produces a small, but significant, increase in jejunal apoA-I synthesis. The data further suggest that apoA-I synthesis in jejunal enterocytes is regulated in part by the availability of lumenal lipid, but that the presence of bile does not exert an additional level of control.(ABSTRACT TRUNCATED AT 400 WORDS)

Human apoA-IV was purified from delipidated urinary chylomicrons. Monospecific antibodies were raised in rabbits and used to develop a double antibody radioimmunoassay (RIA). Displacement of 125I-labeled apoA-IV by plasma or purified chylomicron apoA-IV resulted in parallel displacement curves, indicating that apoA-IV from both sources share common antigenic determinants. The apoA-IV level in plasma from normal healthy fasting male subjects (n = 5) was 37.4 +/- 4.0 mg/dl, while fat-feeding increased the level to 49.1 +/- 7.9 mg/dl (P less than 0.05) at 4 hr. The apoA-IV level in plasma from abetalipoproteinemic fasting subjects was 13.7 +/- 3.1 mg/dl (n = 5). Plasma from a single fasting Tangier subject showed a reduced apoA-IV level of 21.1 mg/dl. The distribution of apoA-IV in fasting and postprandial plasma was determined by 6% agarose gel chromatography. Fifteen to 25% of plasma apoA-IV eluted in the region of plasma high density lipoprotein (HDL), with the remainder eluting in subsequent column fractions. In abetalipoproteinemic plasma this HDL fraction is reduced and lacks apoA-IV, suggesting that at least some of the apoA-IV on these particles is normally derived from triglyceride-rich lipoproteins. Lipemic plasma from a fat-fed subject showed a small rise (3%) in chylomicron-associated apoA-IV. Gel-filtered HDL and subsequent apoA-IV-containing fractions were subjected to 4-30% polyacrylamide gradient gel electrophoresis (4/30 GGE), and apoA-IV was identified by immunolocalization following transfer of proteins to nitrocellulose paper. In normal plasma apoA-IV was localized throughout all HDL fractions. In addition, normal plasma contained apoA-IV localized in a small particle (diameter 7.8-8.0 nm). This particle also contained apoA-I and lipid. A markedly elevated saturated to unsaturated cholesteryl ester ratio was present in gel-filtered plasma fractions containing small HDL, suggesting an intracellular origin of these particles. In abetalipoproteinemic plasma apoA-IV was absent from all HDL fractions except for the small HDL particles, suggesting that they are not derived from the surface of triglyceride-rich particles. All plasmas contained free apoA-IV. In contrast to gel-filtered plasma, lipoprotein subfractions of fasted normal plasma prepared in the ultracentrifuge primarily contained apoA-IV in the d greater than 1.26 g/ml fraction, suggesting an artifactual redistribution of the apolipoprotein during centrifugation. Overall, these data suggest that apoA-IV secretion into plasma is increased with fat feeding, and that apoA-IV normally exists as both a free apolipoprotein and in association with HDL particles

Previous studies have defined forms of high density lipoproteins (HDL) in rat mesenteric lymph, suggesting that they have a secretory origin. This study describes the isolation and characterization of intestinal intracellular HDL. Two preparations were made as follows: (a) Rat enterocytes were isolated and a Golgi organelle fraction was prepared. (b) Cell homogenates were subjected to nitrogen cavitation and a cytoplasmic fraction was prepared. Lipoproteins were isolated from both preparations by sequential ultracentrifugation. When the HDL fraction (1.07-1.21 g/ml) was subjected to isopyknic density gradient ultracentrifugation, a peak of apoproteins A-I and B (apoA-I and apoB, respectively) was found at a density of 1.11-1.14 g/ml. Electron microscopy of the fraction showed spherical particles ranging in size from 6 to 13 nm. Immunoelectrophoresis revealed a precipitin arc in the alpha region against apoA-I which extended into the pre-beta region where a precipitin arc against apoB was also seen. ApoB antisera depleted the pre-beta particles whereas the alpha migrating particles remained. Lipid analysis of the whole HDL fraction revealed phospholipid, cholesteryl ester, and triglyceride as the major lipids. [3H]leucine was then administered into the duodenum and a radiolabeled intracellular HDL fraction was isolated. The newly synthesized apoproteins of the HDL fraction, as determined by gel electrophoresis, were apoB, apoA-I, and apolipoprotein A-IV (ApoA-IV). Immunoprecipitation of the apoB particles revealed apoA-I and apoA-IV in the supernatant. These data demonstrate that there are at least two intracellular intestinal forms of HDL particles, one of which contains apoB. The other particle contains apoA-I and apoA-IV, has alpha mobility, is spherical, and resembles a particle found in the lymph

The primary translation product of human intestinal apolipoprotein A-IV mRNA was purified from ascites and wheat germ cell-free systems. Comparison of its NH2-terminal sequence with mature, chylomicron-associated apo-A-IV revealed that apo-A-IV was initially synthesized with a 20-amino acid long NH2-terminal extension: Met-X-Leu-X-Ala-Val-Val-Leu-X-Leu-Ala-Leu-Val-Ala-Val-Ala-Leu-X-X-Ala. Co-translational cleavage of the cell-free product as well as Edman degradation of the stable intracellular form of the protein recovered from Hep G2 cells indicated that this entire 20-amino acid sequence behaved as a signal peptide. There is at least 55% sequence homology between the rat and human apo-A-IV signal peptides and 33% homology between the human A-I and A-IV presegments. Agarose gel chromatography of Hep G2 culture media indicated that neither apo-A-IV nor -A-I is associated with particles that have physical properties resembling any of the plasma lipoprotein density classes. Incubation of plasma with Hep G2 media resulted in transfer of A-I but not A-IV to lipoproteins. Since the NH2 termini of co-translationally cleaved and chylomicron-associated apo-A-IV are identical, it is apparent that 1) this polypeptide does not undergo NH2-terminal post-translational proteolysis like proapo-A-II or proapo-A-I, and 2) regulation of A-IV-lipoprotein interaction is not dependent on any NH2-terminal proteolytic processing event