Conversion Of Very Low Density Lipoprotein Research Articles

Effects of APOC3 Heterozygous Deficiency on Plasma Lipid and Lipoprotein Metabolism.

Objective- Apo (apolipoprotein) CIII inhibits lipoprotein lipase (LpL)-mediated lipolysis of VLDL (very-low-density lipoprotein) triglyceride (TG) and decreases hepatic uptake of VLDL remnants. The discovery that 5% of Lancaster Old Order Amish are heterozygous for the APOC3 R19X null mutation provided the opportunity to determine the effects of a naturally occurring reduction in apo CIII levels on the metabolism of atherogenic containing lipoproteins. Approach and Results- We conducted stable isotope studies of VLDL-TG and apoB100 in 5 individuals heterozygous for the null mutation APOC3 R19X (CT) and their unaffected (CC) siblings. Fractional clearance rates and production rates of VLDL-TG and apoB100 in VLDL, IDL (intermediate-density lipoprotein), LDL, apo CIII, and apo CII were determined. Affected (CT) individuals had 49% reduction in plasma apo CIII levels compared with CCs ( P<0.01) and reduced plasma levels of TG (35%, P<0.02), VLDL-TG (45%, P<0.02), and VLDL-apoB100 (36%, P<0.05). These changes were because of higher fractional clearance rates of VLDL-TG and VLDL-apoB100 with no differences in production rates. CTs had higher rates of the conversion of VLDL remnants to LDL compared with CCs. In contrast, rates of direct removal of VLDL remnants did not differ between the groups. As a result, the flux of apoB100 from VLDL to LDL was not reduced, and the plasma levels of LDL-cholesterol and LDL-apoB100 were not lower in the CT group. Apo CIII production rate was lower in CTs compared with CCs, whereas apo CII production rate was not different between the 2 groups. The fractional clearance rates of both apo CIII and apo CII were higher in CTs than CCs. Conclusions- These studies demonstrate that 50% reductions in plasma apo CIII, in otherwise healthy subjects, results in a significantly higher rate of conversion of VLDL to LDL, with little effect on direct hepatic uptake of VLDL. When put in the context of studies demonstrating significant protection from cardiovascular events in individuals with loss of function variants in the APOC3 gene, our results provide strong evidence that therapies which increase the efficiency of conversion of VLDL to LDL, thereby reducing remnant concentrations, should reduce the risk of cardiovascular disease.

Human LDL Receptor Enhances Sequestration of ApoE4 and VLDL Remnants on the Surface of Hepatocytes but Not Their Internalization in Mice

In humans, apolipoprotein (apo) E4 is associated with elevated plasma cholesterol levels and a high risk of developing atherosclerosis, whereas apoE2 is protective. Here we investigate the mechanism by which mice expressing human apoE isoforms recapitulate this association when they also express high levels of human low-density lipoprotein receptor (LDLR). Primary hepatocytes from apoE4 mice secreted less apoE into the medium than hepatocytes from apoE2 mice. Increased LDLR expression decreased this secretion and increased degradation of apoE4. An apoE4-GFP fusion protein expressed in the liver of apoE-deficient mice accumulated on the hepatocyte surface bordering the space of Disse in an LDLR-dependent manner. Fluorescence-labeled very low-density lipoprotein (VLDL) remnants accumulated on the hepatocyte surface in apoE4 mice with high LDLR, but they were internalized poorly. In contrast, apoE2-GFP did not accumulate on the hepatocyte surface even when the LDLR expression was high, but apoE2 mice with high LDLR internalized the remnants avidly without sequestering them on the hepatocyte surface. The high affinity of apoE4 to the LDLR enhances VLDL sequestration on the hepatocyte surface but delays their internalization. This delay likely increases VLDL conversion to cholesterol-enriched remnants in apoE4 mice with high LDLR, and probably to LDL in humans with apoE4.

FISH OILS, PHYTOSTEROLS AND WEIGHT LOSS IN THE REGULATION OF LIPOPROTEIN TRANSPORT IN THE METABOLIC SYNDROME: LESSONS FROM STABLE ISOTOPE TRACER STUDIES

1. Dyslipoproteinaemia is a cardinal feature of the metabolic syndrome that accelerates atherosclerosis. It is characterized by high plasma concentrations of triglyceride-rich and apolipoprotein (apo) B-containing lipoproteins, with depressed concentrations of high-density lipoprotein (HDL). Dysregulation of lipoprotein metabolism in these subjects may be due to a combination of overproduction of very-low density lipoprotein (VLDL) apoB-100, decreased catabolism of apoB-containing particles and increased catabolism of HDL apoA-I particles. 2. Nutritional interventions may favourably alter lipoprotein transport in the metabolic syndrome. We review our collaborative studies, using stable isotopes and compartmental modelling, of the kinetic effects of fish oils, plant sterols (phytosterols) and weight reduction on the dyslipoproteinaemia in this disorder. 3. Fish oil supplementation diminished hepatic secretion of VLDL-apoB and enhanced conversion of VLDL to low-density lipoprotein (LDL)-apoB, without altering catabolism. 4. Plant sterols (phytosterols) did not have a significant effect on plasma concentrations of lipids and lipoprotein or the kinetics of apoB and apoA-I. 5. Modest weight reduction optimally decreased plasma triglyceride and LDL-cholesterol via reduction in hepatic apoB secretion and reciprocal upregulation of LDL catabolism. 6. The scope and potential of future studies using stable isotope tracers is discussed.

Metabolism of cholesterol ester of apolipoprotein B100-containing lipoproteins in dogs: evidence for disregarding cholesterol ester transfer.

It has been shown that dogs exhibit no cholesterol ester transfer protein (CETP) activity in vitro, in contrast to humans. The aim of our study was to determine modalities of in vivo plasma cholesterol ester turnover in this species, using a kinetic approach with stable isotopes. Kinetics of very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) were studied in seven adult male Beagle dogs using a dual isotope approach through endogenous labelling of both their cholesterol moiety and their protein moiety. A primed constant infusion of both [1,2(13)C]acetate and [5,5,5-2H3]leucine enabled us to obtain measurable deuterium enrichments by gas chromatography-mass spectrometry for plasma leucine and apoB100, as well as measurable 13C enrichment by gas chromatography-combustion-isotopic ratio mass spectrometry for unesterified cholesterol and cholesterol ester in the VLDL and LDL. Two identical multicompartmental models (SAAM II) were used together for the analysis of tracer kinetics' data of proteins and cholesterol. Characterization of the apoB100-containing lipoprotein cholesterol ester model allowed determination of kinetic parameters of VLDL and LDL cholesterol ester metabolism. We succeeded in modelling VLDL and LDL cholesterol ester metabolism and apoB100 metabolism simultaneously. Fractional catabolic rate (FCR) of apoB100 and CE had the same values. Introducing cholesterol ester transfer between lipoproteins in the model did not significantly improve the fit. Total VLDL FCR was 2.97 +/- 01.47 h(-1). Approximately one-quarter corresponded to the direct removal of VLDL (0.81 +/- 00.34 h(-1)) and the remaining three-quarters corresponded to the fraction of VLDL converted to LDL, which represented a conversion of VLDL into LDL of 2.16 +/- 01.16 h(-1). Low-density lipoproteins were produced exclusively from VLDL conversion and were then removed (0.031 +/- 0.004 h(-1)) from plasma. These kinetic data showed that VLDL cholesterol ester and LDL cholesterol ester metabolism followed VLDL and LDL apoB100 metabolism, and that consequently there is no in vivo transfer of cholesterol ester in dogs.

Ex vivo measurement of lipoprotein lipase-dependent very low density lipoprotein (VLDL)-triglyceride hydrolysis in human VLDL: an alternative to the postheparin assay of lipoprotein lipase activity?

The plasma lipolysis of triglyceride (TG)-rich lipoproteins is mainly due to the activity of lipoprotein lipase (LPL). Albeit important for our analysis of certain physiopathological situations, the determination of the magnitude of LPL-dependent lipolysis is not easy to perform. This essentially results from the binding of LPL to the luminal surface of vascular endothelium. The measurements of the whole putative LPL activity have been achieved after injection of heparin, a procedure that releases LPL from endothelium. However, the physiopathological relevance of this postheparin lipolysis assay (PHLA) remains questionable because it has never been demonstrated that the bulk of endothelium-bound LPL was active. It has been recently shown that a small part of LPL is associated to circulating lipoproteins in nonheparinized plasma, raising the possibility that the lipolysis mediated by this circulating LPL might reflect the overall LPL-dependent TG hydrolysis in plasma. To address this question, we developed a new lipolysis assay in which the very low density lipoprotein (VLDL)-bound LPL-dependent VLDL-TG hydrolysis (LVTH) was directly determined through the measurement of nonesterified fatty acid (NEFA) release during in vitro incubations. LVTH measurements were performed in control subjects, in type 2 diabetics, and in either heterozygous or homozygous LPL-deficient patients. In the latter group, LVTH values were extremely low. Those of heterozygous patients and of diabetics were similarly decreased by about 40% with respect to control group. Plasma TG concentrations exhibited an inverse relationship with LVTH level. In a subgroup of subjects, LVTH and PHLA were positively correlated and the inverse correlation of LVTH with plasma or VLDL-TG concentration was stronger than that obtained with PHLA. To further study the validity of this new assay, we measured LVTH in nine subjects who were studied for their catabolism of VLDL labeled with stable isotope. No relation was observed between the direct hepatic removal of VLDL and LVTH, whereas the latter was strikingly correlated with the rate of conversion of VLDL to intermediary density lipoprotein. Collective consideration of these findings strongly suggests that LVTH is a physiologically relevant index which could advantageously replace the measurements of PHLA in numerous physiopathological situations.

Lipolytic remnants of human VLDL produced in vitro: effect of HDL levels in the lipolysis mixtures on the apoCs to apoE ratio and metabolic properties of VLDL core remnants

To determine the role of high-density lipoprotein (HDL) as an acceptor of lipolytic surface remnants of very low density lipoprotein (VLDL) in the metabolism of VLDL core remnants, we examined the effect of HDL levels in the VLDL lipolysis mixture on 1) the morphology and the apoCs to E ratio in VLDL core remnants and 2) the metabolic properties of VLDL core remnants in human hepatoma cell line HepG2 and human hepatocytes in the primary culture. Normolipidemic VLDL was lipolyzed in vitro by purified bovine milk lipoprotein lipase (LpL) in a lipolysis mixture containing a physiologic level of VLDL and albumin (30 mg VLDL-cholesterol (CH)/dl and 6% albumin) in the absence and presence of either a low HDL level (VLDL-CH:HDL-CH = 3:1) or a high HDL level (VLDL-CH:HDL-CH = 1:4). Lipolysis of VLDL in either the absence or presence of HDL resulted in the hydrolysis of >85% of VLDL-triglycerides (TG) and the conversion of VLDL into smaller and denser particles. In the absence of HDL, heterogeneous spherical particles with numerous surface vesicular materials were produced. In the presence of low or high HDL, spherical particles containing some or no detectable vesicular surface components were produced. The apoCs to apoE ratios, as determined by densitometric scanning of the SDS polyacrylamide gradient gel, were 2.89 in control VLDL and 2.27, 0.91, and 0.22 in VLDL core remnants produced in the absence and in the presence of low and high HDL levels, respectively. In vitro lipolysis of VLDL markedly increased binding to HepG2 cells at 4°C and internalization and degradation by human hepatocytes in primary culture at 37°C. However, the HDL-mediated decrease in the apoCs to apoE ratio had a minimal effect on binding, internalization, and degradation of VLDL core remnants by HepG2 cells and human hepatocytes in primary culture. In order to determine whether HepG2 bound VLDL and VLDL core remnants are deficient in apoCs, 125I-labeled VLDL and VLDL core remnants were added to HepG2 culture medium at 4°C. The bound particles were released by heparin, and the levels of 125I-labeled apoCs and apoE, relative to apoB, in the released particles were examined. When compared with those initially added to culture medium, the VLDL and VLDL core remnants released from HepG2 cells had a markedly increased (113%) level of apoE and a reduced (30–39%), but not absent, level of apoCs. We conclude that apoCs, as a minimum structural and/or functional component of VLDL and VLDL core remnants, may not have an inhibitory effect on the binding of VLDL or VLDL core remnants to hepatic apoE receptors. —Chung, B. H., and N. Dashti. Lipolytic remnants of human VLDL produced in vitro: effect of HDL levels in the lipolysis mixtures on the apoCs to apoE ratio and metabolic properties of VLDL core remnants.

Reduction in visceral adipose tissue is associated with improvement in apolipoprotein B-100 metabolism in obese men.

We investigated the effect of reduction in visceral obesity on the kinetics of apolipoprotein B-100 (apoB) metabolism in a controlled dietary intervention study in 26 obese men. Hepatic secretion of very low density lipoprotein (VLDL) apoB was measured using a primed, constant, infusion of 1-[13C]leucine. In seven men receiving the reduction diet, intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) apoB kinetics were also determined. ApoB isotopic enrichment was measured using gas chromatography-mass spectrometry, and SAAM-II was used to estimate the fractional turnover rates. Subcutaneous and visceral adipose tissues at the L3 vertebra were quantified by magnetic resonance imaging. With weight reduction there was a significant decrease (P < 0.05) in body mass index, waist circumference, and visceral adipose tissue. The plasma concentrations of total cholesterol, triglyceride, insulin, and lathosterol also significantly decreased (P < 0.05). Compared with weight maintenance, weight reduction significantly decreased the VLDL apoB concentration, pool size, and hepatic secretion of VLDL apoB (delta+2.5+/-4.6 vs. delta-14.7+/-4.0 mg/kg fat free mass-day; P = 0.010), but did not significantly alter its fractional catabolism. Weight reduction was also associated with an increased fractional catabolic rate of LDL apoB (0.24+/-0.07 vs. 0.54+/-0.10 pools/day; P = 0.002) and conversion of VLDL to LDL apoB (11.7+/-2.5% vs. 56.3+/-11.4%; P = 0.008). A change in hepatic VLDL apoB secretion was significantly correlated with a change in visceral adipose tissue area (r = 0.59; P = 0.043), but not plasma concentrations of insulin, free fatty acids, or lathosterol. The data support the hypothesis that a reduction in visceral adipose tissue is associated with a decrease in the hepatic secretion of VLDL apoB, and this may be due to a decrease in portal lipid substrate supply. Weight reduction may also increase the fractional catabolism of LDL apoB, but this requires further evaluation.

Regulation of apolipoprotein B-containing lipoproteins by dietary soluble fiber in guinea pigs

Dietary soluble-fiber sources such as pectin, guar gum, or psyllium decrease plasma concentrations of low-density-lipoprotein (LDL) cholesterol in guinea pigs by distinct mechanisms, including increases in LDL apolipoprotein (apo) B turnover and/or decreases in LDL apo B flux (J Lipid Res 1995; 36:2394-404). The present studies were undertaken to test whether changes in the rates of very-low-density lipoprotein (VLDL) apo B secretion, VLDL conversion to LDL, and hepatic uptake of VLDL were related to the cholesterol-lowering actions of these soluble fibers. Guinea pigs were fed (by wt) 12.5% pectin, 12.5% guar gum, 7.5% psyllium, or a control diet containing cellulose as the fiber source. Plasma cholesterol concentrations were significantly lower in guinea pigs fed pectin, guar gum, and psyllium by 42%, 46%, and 35%, respectively (P < 0.001), compared with those animals fed the control diet, whereas plasma triacylglycerol concentrations were lower only with guar gum intake. The secretion rate of triacylglycerol, determined after Triton was injected to block VLDL catabolism, was not different among dietary treatment groups whereas the secretion rate of apo B was lower with pectin, guar gum, and psyllium intakes (P < 0.01). In addition, pectin, guar gum, and psyllium significantly altered the composition of newly secreted VLDLs by increasing the number of triacylglycerol and phospholipid molecules in the secreted lipoprotein, indicating the presence of larger nascent VLDLs. In contrast, the average particle diameter of mature VLDLs as determined by electron microscopy was smaller in the dietary soluble-fiber groups in the following order: pectin < psyllium < guar gum. Plasma lecithin-cholesteryl acyltransferase and cholesteryl ester transfer protein activities were lower with intake of pectin, guar gum, and psyllium (P < 0.01). Injection of radiolabeled lipoproteins indicated that pectin, guar gum, and psyllium intakes resulted in more rapid VLDL and LDL apo B turnover and lower LDL apo B flux in guinea pigs fed guar gum and psyllium. These results suggest that the secretion rate of apo B, compositional changes in lipoproteins occurring in the intravascular compartment, and up-regulation of LDL receptors-associated with faster LDL apo B turnover rates-are secondary metabolic responses induced by dietary fiber that contribute to the lowering of plasma cholesterol concentrations.

Effects of omega-3 fatty acids on intravascular lipolysis of very-low-density lipoproteins in humans

Very-low-density lipoproteins (VLDLs) are the major carriers of fasting plasma triglyceride (TG). TG-enriched VLDLs become cholesterol (C)-enriched low-density lipoproteins (LDLs) through hydrolysis facilitated by lipoprotein lipase (LPL). Omega-3 fatty acid (n-3 FA) supplementation may increase LDL-C while decreasing plasma TG in hypertriglyceridemic patients. It has been proposed that n-3 FAs increase LDL-C by promoting production of TG-poor VLDL and accelerating conversion of VLDL to LDL. To study the effects of n-3 FA supplementation on in vivo lipolysis of VLDL directly, we treated 11 hypertriglyceridemic subjects with n-3 FA (3.3 g/d). Each participant was studied three times: at baseline, after a 1-month period of run-in olive oil placebo, and after 1 more month of n-3 FA supplementation. Lipolysis was induced by intravenous infusion of heparin for 2 hours. Plasma samples were obtained every 30 minutes for determination of lipids and apoproteins (apos), separation of individual lipoproteins by fast protein liquid chromatography (FPLC), and measurement of LPL and hepatic TG lipase (HTGL) levels. n-3 FA supplementation decreased fasting plasma TG (2.51 ± 0.23 v 3.97 ± 0.46 mmol/L), VLDL-TG (1.08 ± 0.18 v 2.35 ± 0.35 mmol/L), and VLDL-C (0.39 ± 0.05 v 0.72 ± 0.13 mmol/L) while increasing LDL-C (3.59 ± 0.21 v 3.00 ± 0.23 mmol/L) and plasma apo B (3.31 ± 0.19 v 2.90 ± 0.17 mmol/L). The absolute rate of TG lipolysis correlated with fasting TG ( r = .74, P < .005) and was lower after n-3 FA supplementation (0.11 ± 0.01 mmol/mL/min) as compared with placebo (0.19 ± 0.01, P < .01), whereas percent decreases from baseline TG levels were similar at entry onto the study (57.4% ± 2.5%), after placebo (58.8% ± 2.7%), and after n-3 FA (52% ± 3.6%). During lipolysis, LDL-C increased in correlation with the decrease in VLDL-C ( r = -.41, P < .03), consistent with the product-precursor relationship. Postheparin LPL activity at 2 hours correlated inversely with fasting TG ( r = −.53, P < .05), but not with either the decreases in VLDL-TG and VLDL-C or the increase in LDL-C. These data demonstrate that n-3 FA supplementation does not accelerate lipolysis of VLDL and even decreases the VLDL-C pool, which is the precursor of LDL-C. Therefore, the increase seen in fasting LDL-C is probably mediated through mechanisms other than acceleration of VLDL lipolysis.

Effects of stigmastanyl-phosphocholine (Ro 16-6532) and lovastatin on lipid and lipoprotein levels and lipoprotein metabolism in the hamster on different diets

Previous studies from our laboratory have shown that oral administration of stigmastanyl-phosphocholine (Ro 16-6532) reduces plasma cholesterol levels in experimental animals on diets free of added cholesterol. In the present study, effects of Ro 16-6532 and lovastatin on lipoprotein levels and metabolism were investigated in male golden Syrian hamsters. In hamsters fed a standard diet, Ro 16-6532 (1 mmol/kg/day) lowered cholesterol in all lipoprotein fractions, as well as apoB-100 and apoA-I. In contrast, lovastatin (25 mumol/kg/day) lowered high density lipoprotein (HDL)-cholesterol but had no effect on low density lipoprotein (LDL)-cholesterol or on apoB-100 or apoA-I while triglycerides and very low density lipoprotein (VLDL)-cholesterol increased. In hamsters fed a coconut fat-supplemented diet, Ro 16-6532 reduced all lipoproteins, with a stronger effect on VLDL- and LDL- than on HDL-cholesterol. Also apoB-100 was reduced. Lovastatin (50 mumol/kg/day) reduced LDL-cholesterol, HDL-cholesterol, and apoA-I while triglycerides and VLDL-cholesterol increased. The drop in LDL-cholesterol seen with both drugs in hamsters fed the diet supplemented with coconut fat occurred without any effect on the plasma removal rate of homologous LDL, or on the content of hepatic LDL-receptors. In contrast, the first phase of removal of homologous radioiodinated VLDL from plasma was markedly increased by both compounds, paralleled with an increased uptake of label in the liver and a decreased appearance of labeled apoB-100 in the LDL-fraction. Furthermore, retinyl ester-labeled chylomicrons were also cleared more rapidly in hamsters treated with Ro 16-6532. Hepatic uptake of label from VLDL and chylomicrons was strongly decreased by pre-injection of lactoferrin. In addition, Ro 16-6532 slightly decreased the secretion rate of VLDL in hamsters fed the coconut fat-supplemented diet. Taken together, these results indicate that the reduction of LDL-cholesterol after treatment with Ro 16-6532 and lovastatin observed in the hamster is mainly due to decreased conversion of VLDL into LDL, consequent to an increased hepatic removal of VLDL remnants. Ro 16-6532 also increased the liver uptake of chylomicron remnants. The hepatic uptake system implicated in this remnant removal can be completely blocked by lactoferrin. The nature of this uptake system is still unknown.

Triglyceride-rich lipoproteins of subjects heterozygous for apolipoprotein E2(Lysl46→Gln) are inefficiently converted to cholesterol-rich lipoproteins

The APOE ∗2(Lys146→Gln) allele behaves like a dominant trait in the expression of familial dysbetalipoproteinemia (FD) (Smit et al., J. Lipid Res. 1990; 31: 42–53). FD patients carrying the APOE ∗2(Lysl46→Gln) allele exhibit less elevated cholesterol to triglyceride ratios in the d < 1.019 g/ml lipoprotein density fraction as compared to classical FD patients displaying homozygosity for the APOE ∗2(Arg158→Cys) allele (0.8 vs. 1.4). Upon treatment of complete serum with lipoprotein lipase (LPL), the mean cholesterol to triglyceride molar ratio of the d < 1.019 g/ml lipoprotein fraction in these FD patients increased only marginally (from 0.8 to 1.1), as compared with that of classical FD subjects (from 1.4 to 2.6) and non-FD control subjects (from 0.7 to 1.5). In order to obtain further evidence for an inefficient lipolysis of the d < 1.019 g/ml lipoprotein fraction in APOE ∗2(Lys146→Gln) carriers, possibly in combination with a less efficient cholesteryl ester transfer protein (CETP) activity, blood samples of APOE ∗2(Lys146→Gln) allele carrying FD patients were analysed and compared with classical FD patients and controls. In the APOE ∗2(Lysl46→Gln) FD patients, the increase in plasma cholesterol was mainly confined to the very low density lipoprotein (VLDL) fraction, whereas in classical FD patients, the levels of cholesterol in the intermediate density lipoprotein (IDL) fraction was also dramatically increased (ratios of VLDL to IDL cholesterol are 4.7 and 2.6, respectively). Family analyses of the APOE ∗2(Lysl46→Gln) FD subjects showed that the apo E to apo B ratio in the d < 1.019 g/ml lipoprotein fraction of allele carriers is 3.5 times as high as that found in non-carriers (2.8 vs. 0.8, by wt.). Also, in the APOE ∗2(Lysl46→Gln) allele carrying family members, the ratio of cholesterol to triglyceride of the d < 1.019 g/ml lipoprotein fraction is less markedly elevated upon addition of LPL when compared to that in non-carrying controls (from 1.1 to 1.8 vs 0.7 to 1.6). The efficiency of the d < 1.019 g/ml lipoprotein fraction of APOE ∗2(Lys146→Gln) FD patients to compete with low density lipoprotein (LDL) for binding to the LDL receptor is intermediate to that of controls and classical APOE ∗2(Arg158→Cys) homozygous FD patients. These findings suggest that in APOE ∗2(Lys146→Gln) allele carriers, the conversion of VLDL into IDL is impaired due to an inefficient lipolysis, possibly in combination with a retarded CETP activity. As a consequence, this might result in a less efficient cellular processing of these lipoproteins via the LDL receptor and in this way explain the dominant behaviour of the APO ∗2(Lys146→Gln) allele in the expression in FD.

Changes in plasma lipoproteins during lipolysis in vivo in the hypertriglyceridemic state

We investigated the effects of the severity of the hypertriglyceridemic state on lipolysis of very-low-density lipoproteins (VLDLs) in vivo. In six patients with mild (Mild, fasting triglyceride 2.54 +/- 0.27 mmol/L) and six with moderate hypertriglyceridemia (Mod, fasting triglyceride 4.63 +/- 0.47 mmol/L), heparin infusion decreased plasma triglycerides in direct correlation with the baseline triglyceride (r = 0.92 in Mild, r = 0.96 in Mod) concentration. Fasting VLDL-triglyceride correlated inversely with postheparin lipoprotein lipase (LPL) (r = -0.85). A decrease in VLDL-triglyceride correlated with baseline VLDL-triglyceride (r = 0.93), but not with postheparin LPL. In the Mild group, low-density-lipoprotein (LDL) cholesterol steadily increased (baseline, 2.90 +/- 0.18 mmol/L; 30 min, 3.03 +/- 0.23 mmol/L; 2 h, 3.15 +/- 0.18 mmol/L) in correlation with the decrease in VLDL-triglyceride (r = 0.89). In the Mod group, LDL cholesterol initially decreased (baseline, 2.51 +/- 0.34 mmol/L; 30 min, 2.30 +/- 0.23 mmol/L) and then increased (2 h, 2.82 +/- 0.28 mmol/L). These results demonstrate a delay in conversion of VLDLs into LDLs in pronounced hypertriglyceridemia, which may contribute to the etiology of low plasma LDL cholesterol.

Overexpression of human lipoprotein lipase in transgenic mice. Resistance to diet-induced hypertriglyceridemia and hypercholesterolemia.

Lipoprotein lipase (LPL) is a key enzyme in the hydrolysis of triglyceride-rich lipoproteins. To determine the role of LPL in lipoprotein metabolism, we established three transgenic mouse lines overexpressing the human LPL gene; the highest expressor line, which transcribed human LPL mRNA in the heart, skeletal muscles, and adipose tissue, was used in this study. The transgenic mice had 5- and 1.7-fold higher LPL activity in adipose tissue and post-heparin plasma, respectively. Plasma triglyceride levels in transgenic mice were 24.2% of that in control mice, and gel filtration chromatography showed that very low density lipoprotein (VLDL) triglycerides were much reduced in transgenic mice. In the chemical analysis of plasma lipoproteins isolated by ultracentrifugation, we found that LDL particles were cholesterol-rich and HDL2 cholesterol was increased 1.4-fold in transgenic mice as compared to control mice. When we injected 125I-VLDL intravenously into transgenic mice, the clearance of 125I-VLDL and the conversion of VLDL to LDL was markedly enhanced as compared to control mice. Furthermore, the clearance of chylomicrons, estimated by both the fat loading and retinyl palmitate loading tests, was significantly enhanced in transgenic mice. After sucrose feeding, no increase in VLDL was observed in transgenic mice. When fed a high cholesterol diet, the development of hypercholesterolemia was suppressed in transgenic mice. These results suggested that LPL determined not only hydrolysis of triglyceride-rich lipoproteins but also lipolytic conversion, and that overexpression of LPL acted to protect against diet-induced hypertriglyceridemia as well as hypercholesterolemia.

Acetaldehyde adducts and autoantibodies against VLDL and LDL in alcoholics

Alcohol consumption markedly increases the hepatic output of very low density lipoprotein (VLDL), whereas it decreases the resulting low density lipoprotein (LDL) levels and apolipoprotein B. As ethylation of apoB-lysine renders LDL immunogenic and accelerates their clearance, and as alcoholics develop antibodies against acetaldehyde-protein adducts, we searched for antibodies against lipoproteins. We measured serum IgG, IgA, and IgM titers against VLDL, LDL and high density lipoprotein (HDL) in 10 non-alcoholics and 35 recently drinking alcoholics by ELISA assay. Alcoholics had higher IgG titers than non-alcoholics against VLDL and LDL; these were higher with VLDL than LDL or HDL. Using VLDL and LDL (but not HDL) from alcoholics gave the greatest response. There was no difference in IgA and IgM reactivity. To search for acetaldehyde adducts, we measured the reactivity of VLDL, LDL, HDL, and residual serum proteins against a rabbit anti P4502E1-acetaldehyde adduct IgG, which recognizes the adducts but not the unmodified proteins (except for P4502E1). ApoB-containing lipoproteins from alcoholics (and to a lesser extent non-lipoprotein proteins) reacted with anti-adduct IgG more strongly than those of non-alcoholics. The difference was striking for VLDL, less for LDL, and not detectable for HDL. This suggests that acetaldehyde reacts with apoB prior to its secretion from the liver and that the altered VLDL are partially removed prior to their conversion to LDL. In conclusion, alcoholics develop acetaldehyde adducts in apoB-containing lipoproteins, particularly VLDL. The immune response to these neoantigens could result in accelerated clearance of VLDL and LDL and decreased conversion of VLDL to LDL.

Low density lipoprotein heterogeneity in spontaneously hypercholesterolemic pigs

We previously described a strain of spontaneously hypercholesterolemic pigs carrying an apo-B allele termed Lpb5. Lpb5 pigs are heterogeneous with respect to the severity of their hypercholesterolemia. We have termed Lpb5 pigs with severe hypercholesterolemia Lpb 5.1 pigs, and those with moderate hypercholesterolemia Lpb 5.2, Lpb 5.1 animals have a dramatic increase in buoyant LDL relative to dense LDL, with a buoyant-to-dense LDL ratio of 2.2. In contrast, Lpb 5.2 and control pigs have buoyant-to-dense LDL ratios of 0.7 and 0.5 respectively. This ratio appears to be a stable characteristic of the Lpb 5.1 phenotype because sexually mature boars have a dramatic decrease in total plasma cholesterol concentration with no decrease in their ratio of buoyant-to-dense LDL. We have previously demonstrated a fourteen-fold overproduction of buoyant LDL in the Lpb 5.1 pigs, with very little conversion of dense LDL to buoyant LDL. In the current work, very low density lipoprotein (VLDL) turnover experiments were conducted to determine whether VLDL conversion to buoyant LDL was increased in the Lpb 5.1 pigs. VLDL conversion to buoyant LDL could not account for the increased production of buoyant LDL in Lpb 5.1 pigs. Thus, we cannot account for the increased production of buoyant LDL in the Lpb 5.1 pigs from any measurable plasma lipoprotein source. We have therefore termed this production of buoyant LDL in the Lpb 5.1 pigs direct buoyant LDL production.

Apolipoprotein E and lipoprotein lipase secreted from human monocyte-derived macrophages modulate very low density lipoprotein uptake.

We investigated the roles of lipoprotein lipase and apolipoprotein E (apoE) secreted from human monocyte-derived macrophages in the uptake of very low density lipoproteins (VLDL). ApoCII-deficient VLDL were isolated from a patient with apoCII deficiency. The lipolytic conversion to higher density and the degradation of the apoCII-deficient VLDL by macrophages were very slight, whereas the addition of apoCII enhanced both their conversion and degradation. This suggests that the lipolysis and subsequent conversion of VLDL to lipoproteins of higher density are essential for the VLDL uptake by macrophages. VLDL incubated with macrophages obtained from subjects with E3/3 phenotype (E3/3-macrophages) showed a 17-fold greater affinity in inhibiting the binding of 2 micrograms/ml 125I-low density lipoprotein (LDL) to fibroblasts than native VLDL, whereas the incubation of VLDL with macrophages obtained from a subject with E2/2 phenotype (E2/2-macrophages) did not cause any increase in their affinity. Furthermore, 3 micrograms/ml 125I-VLDL obtained from a subject with E3/3 phenotype were degraded by E3/3-macrophages to a greater extent than by E2/2-macrophages (2-fold), indicating that VLDL uptake is influenced by the phenotype of apoE secreted by macrophages. From these results, we conclude that both lipolysis by lipoprotein lipase and incorporation of apoE secreted from macrophages alter the affinity of VLDL for the LDL receptors on the cells, resulting in facilitation of their receptor-mediated endocytosis.

Structural Domains of Human Apolipoprotein B-100: Differential Accessibility to Limited Proteolysis of B-100 in Low Density and Very Low Density Lipoproteins

The structural domains of human apolipoprotein B-100 in low density lipoproteins (LDL) and the conformational changes of B-100 that accompany the conversion of very low density lipoproteins (VLDL) to LDL were investigated by limited proteolysis with 12 endoproteases of various specificities, and their cleavage sites were determined. In B-100 of LDL, we identified two peptide regions that are highly susceptible to proteolytic cleavage. One region encompassed about 40 amino acids (residues 1280-1320, designated as the NH2-terminal region) and the other about 100 amino acids (residues 3180-3280, designated as the COOH-terminal region). IN LDL, the cleavage sites in both susceptible regions of B-100 were readily accessible to limited proteolysis; but in VLDL, only sites in the COOH-terminal region were readily accessible. Moreover, B-100 in VLDL appeared less degraded than B-100 in LDL by all enzymes used. Reduction of disulfide bonds of B-100 in both LDL and VLDL before digestion by Staphylococcus aureus V8 protease and clostripain exposed additional cleavage sites and increased the rate of B-100 degradation, suggesting that disulfide bonds probably exert conformational constraints. These results indicate the presence of three principal structural domains in B-100 of LDL that are relatively resistant to limited proteolysis. These three domains are connected by the two susceptible peptide regions. Our results also demonstrate differential accessibility of cleavage sites in B-100 of LDL and VLDL to limited proteolysis. This differential accessibility suggests that substantial changes in the conformation or environment of B-100 accompany the conversion of VLDL to LDL.

Lipoprotein metabolism during acute inhibition of lipoprotein lipase in the cynomolgus monkey.

To clarify the role of lipoprotein lipase (LPL) in the catabolism of nascent and circulating very low density lipoproteins (VLDL) and in the conversion of VLDL to low density lipoproteins (LDL), studies were performed in which LPL activity was inhibited in the cynomolgus monkey by intravenous infusion of inhibitory polyclonal or monoclonal antibodies. Inhibition of LPL activity resulted in a three- to fivefold increase in plasma triglyceride levels within 3 h. Analytical ultracentrifugation and gradient gel electrophoresis demonstrated an increase predominantly in more buoyant, larger VLDL (Sf 400-60). LDL and high density lipoprotein (HDL) cholesterol levels fell during this same time period, whereas triglyceride in LDL and HDL increased. Kinetic studies, utilizing radiolabeled human VLDL, demonstrated that LPL inhibition resulted in a marked decrease in the catabolism of large (Sf 400-100) VLDL apolipoprotein B (apoB). The catabolism of more dense VLDL (Sf 60-20) was also inhibited, although to a lesser extent. However, there was a complete block in the conversion of tracer in both Sf 400-100 and 60-20 VLDL apoB into LDL during LPL inhibition. Similarly, endogenous labeling of VLDL using [3H]leucine demonstrated that in the absence of LPL, no radiolabeled apoB appeared in LDL. We conclude that although catabolism of dense VLDL continues in the absence of LPL, this enzyme is required for the generation of LDL.

The catabolism of very low density lipoproteins.

The lipolysis of very low density lipoproteins (VLDL) in vitro is a useful model for the study of the process of conversion of this triacylglycerol rich lipoprotein into low (LDL) and high (HDL) density lipoproteins. Data is reviewed to show that a portion of surface cholesterol and phospholipid which becomes redundant during lipolysis is lost from the lipoprotein. In the absence of HDL, the material forms lipoprotein-X (LpX) like vesicles which are not readily disrupted by HDL once they are formed. In the presence of HDL during lipolysis, the material is largely incorporated into HDL. The data is used to suggest a mechanism of formation of LpX-like vesicles in conditions where the production of surface remnants exceeds the capacity of HDL to disrupt them. Evidence is also provided to show that apolipoproteins (apo) C-II, C-III, and E are lost from VLDL and that this loss is primarily in association with a neutral core particle of HDL size which can subsequently exchange lipids and apolipoproteins with plasma HDL. Such a mechanism could account for the removal of apo E and excess cholesteryl ester which is necessary for conversion of VLDL to LDL. The role of hepatic lipase in this process remains speculative. Recent evidence is reviewed and used to propose that the enzyme may serve to rearrange the neutral core and surface composition of LDL and HDL subfractions to allow for the packaging of cholesteryl esters and the cycling of apolipoproteins.

Abnormalities in very low, low and high density lipoproteins in hypertriglyceridemia. Reversal toward normal with bezafibrate treatment.

The effects of triglyceridemia on plasma lipoproteins were investigated in 16 hypertriglyceridemic (HTG) subjects (222-2,500 mg/dl) before and after the initiation of bezafibrate therapy. Bezafibrate caused a mean reduction of 56% in plasma triglyceride and increased the levels of lipoprotein and hepatic triglyceride lipases by 260 and 213%, respectively. The natures of very low density lipoprotein (VLDL), isolated at plasma density and of low and high density lipoprotein (LDL and HDL), separated by zonal ultracentrifugation, were determined. HTG-LDL appears as multiple fractions whereas HTG-HDL is seen predominantly as HDL3. HTG-VLDL is relatively poor in apoproteins and triglycerides but enriched in free and esterified cholesterol. HTG-LDL (main fraction) is depleted of free and esterified cholesterol but enriched in apoprotein and triglyceride. It is also denser and smaller than normal. HTG-HDL3 is denser than N-HDL3 and demonstrates compositional abnormalities similar to those of HTG-LDL. With the reduction of the VLDL mass, all abnormalities revert towards normal. This is accompanied by an increase in LDL-apoprotein B and cholesterol levels, which indicates an increased conversion of VLDL to LDL. Significant correlations between plasma triglyceride and the degree of all abnormalities are shown. The data obtained during treatment corroborate these relationships. The observations support the concept that most abnormalities reflect the degree of triglyceridemia. We suggest that plasma core-lipid transfer protein(s) is an effector of the abnormal cholesteryl ester distribution. Its prolonged action on increasingly large and slowly metabolized VLDL populations would entail a correspondingly excessive transfer of cholesteryl ester to VLDL and of triglyceride to LDL and HDL. It is calculated that, in moderate HTG, LDL and HDL contain only 50% of the normal cholesterol load. It is suggested that cholesteryl ester redistribution in HTG might be important in regulating metabolic events.