Dorothy Bedford

Metabolic Basis of Hypotriglyceridemic Effects of Insulin in Normal Men

The mechanism by which acute insulin administration alters VLDL apolipoprotein (apo) B subclass metabolism and thus plasma triglyceride concentration was evaluated in 7 normolipidemic healthy men on two occasions, during a saline infusion and during an 8.5-hour euglycemic hyperinsulinemic clamp (serum insulin, 490 +/- 30 pmol/L). During the insulin infusion, plasma triglycerides decreased by 22% (P < .05), and serum free fatty acid decreased by 85% (P < .05). The plasma concentration of VLDL1 apo B fell 32% during the insulin infusion, while that of VLDL2 apo B remained constant. A bolus injection of [3-(2)H]leucine was given on both occasions to trace apo B kinetics in the VLDL1 and VLDL2 subclasses (Svedberg flotation rate, 60-400 and 20-60, respectively), and the kinetic basis for the change in VLDL levels caused by insulin was examined using a non-steady-state multicompartmental model. The mean rate of VLDL1 apo B synthesis decreased significantly by 35% (P < .05) after 0.5 hour of the insulin infusion (523 +/- 87 mg/d) compared with the saline infusion (808 +/- 91 mg/d). This parameter was allowed to vary with time to explain the fall in VLDL1 concentration. After 8.5 hours of hyperinsulinemia, the rate of VLDL1 apo B synthesis was 51% lower (321 +/- 105 mg/d) than during the saline infusion (651 +/- 81 mg/d, P < .05). VLDL2 apo B production was similar during the saline (269 +/- 35 mg/d) and insulin (265 +/- 37 mg/d) infusions. No significant changes were observed in the fractional catabolic rates of either VLDL1 or VLDL2 apo B. We conclude that acute hyperinsulinemia lowers plasma triglyceride and VLDL levels principally by suppressing VLDL1 apo B production but has no effect on VLDL2 apo B production. These findings indicate that the rates of VLDL1 and VLDL2 apo B production in the liver are independently regulated.

Influence of apolipoprotein E polymorphism on apolipoprotein B-100 metabolism in normolipemic subjects.

This study examined apolipoprotein (apo) B metabolism in normolipemic subjects homozygous for the apo E2 (n = 4), apo E3 (n = 5), or apo E4 (n = 5) phenotype. Radioiodinated very low density lipoprotein (VLDL1) (ultracentrifuge flotation rate [Sf] 60-400) and VLDL2 (Sf 20-60) were injected into volunteers and the conversion of apo B was followed through intermediate density lipoprotein (IDL) to low density lipoprotein (LDL). Subjects homozygous for E3 converted approximately 50% of LVDL2 to LDL, the remainder being lost by direct catabolism. Those with the E2 phenotype produced less VLDL1, but converted more of it to VLDL2 (compared to E3 subjects). They displayed a characteristic dyslipidemia with the presence of slowly catabolized VLDL1 and VLDL2 remnants. LDL levels were low owing to increased direct catabolism of VLDL2 and IDL and a reduced efficiency of delipidation; only 25% of VLDL2 apo B was directed to LDL production. In contrast, E4 subjects converted more VLDL2 apo B to LDL than E3 subjects. About 70% of VLDL2 apo B was found in LDL; direct catabolism of VLDL and IDL was reduced as was the fractional catabolic rate of LDL (0.2 vs. 0.26 in E3 subjects). These changes in the VLDL----IDL----LDL metabolic cascade can in part be explained by alterations in hepatic LDL receptors with E2 subjects having higher and E4 subjects lower activities than those in E3 homozygotes.

Effect of heparin-stimulated plasma lipolytic activity on VLDL APO B subclass metabolism in normal subjects

Heparin given intravenously enhances lipolysis, although fasting lipids are not markedly altered in long-term administration. In the present study we investigated heparin-induced acute perturbation of VLDL subclass metabolism. Eight men were examined during a control study and during an 8.5 h infusion of heparin. 2H3-leucine was used as tracer and kinetic constants derived using a non-steady-state model. Heparin infusion increased both plasma lipoprotein and hepatic lipase activity and raised plasma FFAs two-fold (P < 0.001). The fractional catabolic rate (FCR) of VLDL1 apo B increased on heparin (25.7 +/- 4.2 and 10.8 +/- 1.7 pools/d, heparin vs. control, P < 0.02). The FCR of VLDL2 apo B increased to 12.6 +/- 1.9 pools/d on heparin vs. 8.8 +/- 1.1 pools/d during the control (NS). Total VLDL apo B production was not significantly changed (824 +/- 45 and 692 +/- 91 mg/d, heparin vs. control, NS). We conclude that during heparin infusion, the catabolism of especially large triglyceride-rich VLDL1 apo B is greatly increased. However, although the FFA levels were high during the heparin study, the production of total VLDL apo B did not rise. These findings are consistent with the known action of heparin on lipoprotein lipase but indicate that acute increase in plasma FFA levels does not lead to a rise in VLDL apo B production.

Ultracentrifugal subfractionation of high-density lipoprotein

Knowledge of the structure, function and metabolism of the plasma lipoproteins has been greatly facilitated by refinement of the high-performance ultracentrifuge. Techniques based on anglehead rotors are widely used to isolate different fractions by increasing in a stepwise fashion the plasma solvent density. This simple approach may, for various reasons, result in the selection of inappropriate density intervals and so alternative zonal procedures have been developed that yield a continuous lipoprotein spectral distribution. However, the limited analytical capacity of this kind of system has highlighted the need for more rapid microcomputer-controlled ultracentrifugation techniques. The merits of each of these approaches are discussed with reference to the subfractionation of high-density lipoproteins.

Moderate Exercise Increases Affinity of Large Very Low-Density Lipoproteins for Hydrolysis by Lipoprotein Lipase.

CONTEXT Postprandial triglyceride (TG) concentration is independently associated with cardiovascular disease risk. Exercise reduces postprandial TG concentrations, but the mechanisms responsible are unclear. OBJECTIVE The objective was to determine the effects of exercise on affinity of chylomicrons, large very low-density lipoproteins (VLDL1), and smaller VLDL (VLDL2) for lipoprotein lipase (LPL)-mediated TG hydrolysis. DESIGN This was designed as a within-participant crossover study. SETTING The setting was a university metabolic investigation unit. PARTICIPANTS Participants were 10 overweight/obese men. INTERVENTIONS Participants undertook two oral fat tolerance tests, separated by 7-14 days, in which they had blood taken while fasting and for 4 hours after a high-fat mixed meal. On the afternoon before one test, they performed a 90-minute treadmill walk at 50% maximal oxygen uptake (exercise trial [EX]); no exercise was performed before the control trial (CON). MAIN OUTCOME MEASURES We measured circulating TG-rich lipoprotein concentrations and affinity of chylomicrons, VLDL1, and VLDL2 for LPL-mediated TG hydrolysis. RESULTS Exercise significantly reduced fasting VLDL1-TG concentration (CON, 0.49 [0.33-0.72] mmol.L(-1); EX, 0.36 [0.22-0.59] mmol.L(-1); geometric means [95% confidence interval]; P = .04). Time-averaged postprandial chylomicron-TG (CON, 0.55 ± 0.10 mmol.L(-1); EX, 0.39 ± 0.08 mmol.L(-1); mean ± SEM; P = .03) and VLDL1-TG (CON, 0.85 ± 0.13 mmol.L(-1); EX, 0.66 ± 0.10 mmol.L(-1); P = .01) concentrations were both lower in EX than CON. Affinity of VLDL1 for LPL-mediated TG hydrolysis increased by 2.2 (1.3-3.7)-fold [geometric mean (95% confidence interval)] (P = .02) in the fasted state and 2.6 (1.8-2.6)-fold (P = .001) postprandially. Affinity of chylomicrons and VLDL2 was not significantly different between trials. CONCLUSIONS Exercise increases affinity of VLDL1 for LPL-mediated TG hydrolysis both fasting and postprandially. This mechanism is likely to contribute to the TG-lowering effect of exercise.

An increased incidence of apolipoprotein E2/E2 and E4/E4 in retinitis pigmentosa

Previous studies from our laboratory have shown that retinitis pigmentosa (RP), a family of hereditary retinal degenerations, is often accompanied by abnormal levels of cholesterol or polyunsaturated fatty acids. The requirement of the retina for n−3 fatty acids is well known, and a defect in the supply of these lipids (e.g., by apolipoproteins) could affect the course of the disease. The present study confirms and extends a report on apolipoprotein E (apo E) isoforms in German RP patients [Jahn, Oette, Esser, Bergmann, and Leiss, (1987)Ophthalmic Res. 19, 285–288] which showed a tenfold increased frequency of the E2/E2 phenotype compared to the average German population. In our study, apo E phenotypes were determined in the probands of 100 Scottish RP families. The findings revealed a 4-fold increase in the incidence of E2/E2 and an 8-fold increase in E4/E4 compared to a Scottish control population. These increases were statistically significant at theP<0.05 andP<0.01 levels, respectively. To investigate the possibility that some of these apparent E2/E2 or E4/E4 phenotypes might actually be new apo E mutations, we examined the behavior of the apo E on sodium dodecyl sulfate-polyacrylamide gels (E2 migrates anomalously) and on isoelectric focusing gels following cysteamine modification of cysteines. These studies showed that two RP patients possibly had new apo E mutations, though amino-terminal sequence analysis revealed no changes in the sequence of the first 19 residues; further sequence analysis is obviously warranted.

Electrophoretic mobility in agarose of very low density lipoprotein subfractions in type III hyperlipoproteinaemia.

The electrophoretic mobilities in agarose gel of the very low density lipoprotein (VLDL) subfractions of Sf greater than 100, 60-100 and 20-60 from six subjects with type III hyperlipoproteinaemia (HLP) have been compared with those from eight normal volunteers. In type III HLP beta or near-beta (slower VLDL) electrophoretic mobility was not necessarily confined to the VLDL fraction of Sf 20-60 in which it may normally be detected.

The composition of low (LDL) and very low (VLDL) density lipoprotein subfractions in type III hyperlipoproteinaemia: Comparison with normal subjects

The lipid and protein composition of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) subfractions (Sf greater than 100, 60--100 and 20--60 VLDL and Sf 10.4--20, 5.7--12 and 3.5--6.5 LDL) in six subjects with type III hyperlipoproteinaemia (HLP) was compared to that of 12 normal subjects. In type III HLP all VLDL subfractions contained increased concentrations of cholesterol and triglycerides and were relatively enriched in cholesterol. VLDL of Sf 20--60 also contained and increased concentration of B-protein. The tetramethylurea (TMU) soluble apolipoproteins of the VLDL subfractions were separated by polyacrylamide disc gel electrophoresis. In the subjects with type III HLP the proportion of arginine rich protein (ARP) was increased in all subfractions. The concentrations of cholesterol and triglycerides were increased in the LDL subfraction of Sf 10.4--20 and cholesterol was decreased in LDL of Sf 5.7--12, but the ratios of cholesterol to triglycerides were not significantly different from those in the LDL subfractions of the normal subjects and the protein composition was also similar. These results provide further evidence that in type III HLP abnormalities are not confined to the stage of conversion of VLDL to LDL, but occur throughout the VLDL spectrum.

Effect of clofibrate on the composition of very low and low density lipoprotein subfractions in type III hyperlipoproteinaemia

The effect of clofibrate on the lipid and protein composition of very low and low density lipoprotein subfractions (VLDL of SF greater than 100, 60-100 and 20-60; LDL of Sf 10.4-20, 5.7-12 and 3.5-6.5), was investigated in 6 patients with type III hyperlipoproteinaemia (HLP). After four weeks of therapy significant reductions occurred in the concentration of cholesterol in each VLDL fraction, and of triglycerides in Sf greater than 100 and Sf 60-100 VLDL. No changes were found in the concentrations of apolipoprotein B or of the total tetramethylurea (TMU) soluble proteins, but in four patients in whom polyacrylamide disc gel electrophoresis of the TMU soluble proteins was carried out, it was found that arginine-rich peptide (ARP) had largely disappeared on therapy. These findings would be in keeping with increased catabolism of VLDL in response to clofibrate. No significant changes were observed in LDL lipid or protein concentrations.

Genetic Determinants of Apolipoprotein B Metabolism

In this thesis the influence of genetic factors on the apolipoprotein B metabolism in humans was investigated. The phenotype of the apolipoprotein E polymorphism was determined for normolipidaemic subjects (n = 1600). The metabolism of apolipoprotein B in fifteen subjects, homozygous for apoE3, apoE4 or apoE2, was examined by VLDL-turnover studies, using trace-labelled VLDL1 (Sf 60-400) and VLDL2 (Sf 20-60). Results were used for computer modelling of the apoB metabolism, which enabled quantitative comparisons between the three study groups. In apoE2/2 subjects, clearance of VLDL1 and VLDL2 as well as transfer from IDL into LDL was found to be delayed and in apoE4/4 subjects the LDL-FCR was reduced as compared to apoE3/3 normolipidaemics. These observations explain the correlation between apoE phenotypes and plasma cholesterol levels, which had been observed previously by others and were confirmed in the present study. The Xbal restriction site polymorphism of the apoB gene was analysed in nineteen hypercholesterolaemic patients and correlated with fractional catabolic rates for LDL as defined by LDL-turnover studies. The X2 allele was found to be linked with a decreased LDL-FCR, in line with previous reports of a correlation between X2X2 genotype and increased plasma cholesterol concentrations. In addition to these studies of common genetic determinants of apoB metabolism, five patients with rare inherited disorders of lipoprotein metabolism were investigated. These conditions were homozygous familial hypercholesterolaemia, lipoprotein lipase deficiency and hepatic lipase deficiency. VLDL-turnovers in these subjects revealed the significance of the LDL-receptor and the two lipolytic enzymes for apolipoprotein B metabolism. Finally, some conclusions were drawn about metabolic heterogeneity within the VLDL subfraction and about apoB synthesis.