Insulin-mediated modifications of myocardial lipoprotein lipase and lipoprotein metabolism

Recirculating organ perfusion in vitro was conducted with hearts from control rats, animals given a single dose of streptozotocin (65 mg/kg) 48 h earlier, and streptozotocin-treated rats administered insulin (5 units), 2 h prior to organ perfusion. During 45-min perfusions, the lipolysis of very low density lipoprotein (VLDL) triglyceride was significantly less in hearts from diabetics than in controls (41.9 +/- 7.3% of control). This was associated with significant reductions in heparin-releasable (functional) lipoprotein lipase and tissue lipoprotein lipase of perfused hearts. The decreases in VLDL triglyceride metabolism and the levels of myocardial lipoprotein lipase were completely reversed by treatment of diabetic rats with insulin 2 h prior to study. Similar improvement of VLDL triglyceride metabolism and increases in myocardial lipoprotein lipase activity were observed in hearts from diabetic rats by direct addition of 100 milliunits/ml of insulin to the recirculating perfusion media. Under these conditions, the increase in both fractions of lipoprotein lipase in response to insulin was completely inhibited, and utilization of VLDL triglyceride was partially inhibited by pre-perfusion with cycloheximide for 10 min. The data derived from either VLDL triglyceride lipolysis in organ perfusion or direct measurement of myocardial lipoprotein lipase demonstrate a direct effect of insulin on myocardial lipoprotein lipase activity, and suggest that the response to insulin may be due in part to effects on protein synthesis.     

Linkage of low-density lipoprotein size to the lipoprotein lipase gene in heterozygous lipoprotein lipase deficiency.

Small low-density lipoprotein (LDL) particles are a genetically influenced coronary disease risk factor. Lipoprotein lipase (LpL) is a rate-limiting enzyme in the formation of LDL particles. The current study examined genetic linkage of LDL particle size to the LpL gene in five families with structural mutations in the LpL gene. LDL particle size was smaller among the heterozygous subjects, compared with controls. Among heterozygous subjects, 44% were classified as affected by LDL subclass phenotype B, compared with 8% of normal family members. Plasma triglyceride levels were significantly higher, and high-density lipoprotein cholesterol (HDL-C) levels were lower, in heterozygous subjects, compared with normal subjects, after age and sex adjustment. A highly significant LOD score of 6.24 at straight theta=0 was obtained for linkage of LDL particle size to the LpL gene, after adjustment of LDL particle size for within-genotype variance resulting from triglyceride and HDL-C. Failure to adjust for this variance led to only a modest positive LOD score of 1.54 at straight theta=0. Classifying small LDL particles as a qualitative trait (LDL subclass phenotype B) provided only suggestive evidence for linkage to the LpL gene (LOD=1. 65 at straight theta=0). Thus, use of the quantitative trait adjusted for within-genotype variance, resulting from physiologic covariates, was crucial for detection of significant evidence of linkage in this study. These results indicate that heterozygous LpL deficiency may be one cause of small LDL particles and may provide a potential mechanism for the increase in coronary disease seen in heterozygous LpL deficiency. This study also demonstrates a successful strategy of genotypic specific adjustment of complex traits in mapping a quantitative trait locus.     

High-density lipoprotein inhibits the oxidative modification of low-density lipoprotein

Oxidatively modified low-density lipoprotein (LDL), generated as a result of incubation of LDL with specific cells (e.g., endothelial cells, EC) or redox metals like copper, has been suggested to be an atherogenic form of LDL. Epidemiological evidence suggests that higher concentrations of plasma high-density lipoprotein (HDL) are protective against the disease. The effect of HDL on the generation of the oxidatively modified LDL is described in the current study. Incubation of HDL with endothelial cells, or with copper, produced much lower amounts of thiobarbituric acid-reactive products (TBARS) as compared to incubations that contained LDL at equal protein concentrations. Such incubations also did not result in an enhanced degradation of the incubated HDL by macrophages in contrast to similarly incubated LDL. On the other hand, inclusion of HDL in the incubations that contained labeled LDL had a profound inhibitory effect on the subsequent degradation of the incubated LDL by the macrophages while having no effect on the generation of TBARS or the formation of conjugated dienes. This inhibition was not due to the modification of HDL as suggested by the following findings. (A) There was no enhanced macrophage degradation of the HDL incubated with EC or copper alone, together with LDL, despite an increased generation of TBARS. (B) HDL with the lysine groups blocked (acetyl HDL, malondialdehyde (MDA) HDL) was still able to prevent the modification of LDL and (C) acetyl HDL and MDA-HDL competed poorly for the degradation of oxidatively modified LDL. It is suggested that HDL may play a protective role in atherogenesis by preventing the generation of an oxidatively modified LDL. The mechanism of action of HDL may involve exchange of lipid peroxidation products between the lipoproteins.     

Activation of lipoprotein lipase by lipoprotein fractions of human serum

Triglycerides in fat emulsions are hydrolyzed by lipoprotein lipase only when they are "activated" by serum lipoproteins. The contribution of different lipoprotein fractions to hydrolysis of triglycerides in soybean oil emulsion was assessed by determining the quantity of lipoprotein fraction required to give half-maximal hydrolysis. Most of the activator property of whole serum from normolipidemic, postabsorptive subjects was in high density lipoproteins. Low density lipoproteins and serum from which all lipoprotein classes were removed had little or no activity. Also, little activator was present in guinea pig serum or in very low density poor serum from an individual with lecithin:cholesterol acyltransferase deficiency, both of which are deficient in high density lipoproteins. Human very low density lipoproteins are potent activators and are much more active than predicted from their content of high density lipoprotein-protein. Per unit weight of protein, very low density lipoproteins had 13 times the activity of high density lipoproteins. These observations suggest that one or more of the major apoproteins of very low density lipoproteins, present as a minor constituent of high density lipoproteins, may be required for the activation process.     

Intestinal lipoprotein assembly

PURPOSE OF REVIEW: The assembly of intestinal lipoproteins is critical for the transport of fat and fat-soluble vitamins. In this review we propose a nomenclature for these lipoproteins and have summarized recent data about their intracellular assembly and factors that modulate their secretion. RECENT FINDINGS: The assembly and secretion of intestinal lipoproteins increases with the augmented synthesis of apoB, apoAIV and lipids. Chylomicron assembly begins with the formation of primordial, phospholipid-rich particles in the membrane, and their conversion to large chylomicrons occurs in the lumen of the smooth endoplasmic reticulum. Chylomicrons are transported from the endoplasmic reticulum via specialized vesicles to the Golgi for secretion. The identification of genetic mutations in chylomicron retention disease indicates that Sar1b may play a critical role in this process. In addition to chylomicron assembly, intestinal cells have been shown to transport dietary cholesterol via apoB-independent pathways, such as efflux. SUMMARY: Understanding the mechanisms involved in the intracellular transport of chylomicrons and chylomicron-independent secretion pathways are expected to be the next frontiers in the field of intestinal lipoprotein assembly and secretion.     

High density lipoprotein metabolism in low density lipoprotein receptor-deficient mice

The LDL receptor (LDLR) and scavenger receptor class B type I (SR-BI) play physiological roles in LDL and HDL metabolism in vivo. In this study, we explored HDL metabolism in LDLR-deficient mice in comparison with WT littermates. Murine HDL was radiolabeled in the protein (¹²⁵I) and in the cholesteryl ester (CE) moiety ([³H]). The metabolism of ¹²⁵I-/[³H]HDL was investigated in plasma and in tissues of mice and in murine hepatocytes. In WT mice, liver and adrenals selectively take up HDL-associated CE ([³H]). In contrast, in LDLR^−/− mice, selective HDL CE uptake is significantly reduced in liver and adrenals. In hepatocytes isolated from LDLR^−/− mice, selective HDL CE uptake is substantially diminished compared with WT liver cells. Hepatic and adrenal protein expression of lipoprotein receptors SR-BI, cluster of differentiation 36 (CD36), and LDL receptor-related protein 1 (LRP1) was analyzed by immunoblots. The respective protein levels were identical both in hepatic and adrenal membranes prepared from WT or from LDLR^−/− mice. In summary, an LDLR deficiency substantially decreases selective HDL CE uptake by liver and adrenals. This decrease is independent from regulation of receptor proteins like SR-BI, CD36, and LRP1. Thus, LDLR expression has a substantial impact on both HDL and LDL metabolism in mice.     

Electronegative low-density lipoprotein

PURPOSE OF REVIEW: The occurrence in blood of an electronegatively charged LDL was described in 1988. During the 1990s reports studying electronegative LDL (LDL(-)) were scant and its atherogenic role controversial. Nevertheless, recent reports have provided new evidence on a putative atherogenic role of LDL(-). This review focuses on and discusses these new findings. RECENT FINDINGS: In recent years, LDL(-) has been found to be involved in several atherogenic features through its action on cultured endothelial cells. LDL(-) induces the production of chemokines, such as IL-8 and monocyte chemotactic protein 1, and increases tumor necrosis factor-alpha-induced production of vascular cell adhesion molecule 1, with these molecules being involved in early phases of leukocyte recruitment. LDL(-) from familial hypercholesterolemic patients also decreases DNA synthesis and intracellular fibroblast growth factor 2 production, which may contribute to impaired angiogenesis and increased apoptosis. In addition, the preferential association of platelet-activating factor acetylhydrolase with LDL(-) has been reported, suggesting a proinflammatory role of this enzyme in LDL(-). SUMMARY: Recent findings suggest that LDL(-) could contribute to atherogenesis via several mechanisms, including proinflammatory, proapoptotic and anti-angiogenesis properties. Further studies are required to define the role of LDL(-) in atherogenesis more precisely and to clarify mechanisms involved in endothelial cell activation.     

Inhibition of human and rat lipoprotein lipase by high-density lipoprotein

The hydrolysis in vitro of preactivated Intralipid (an artificial triacylglycerol-phospholipid emulsion) by rat adipose tissue lipoprotein lipase is inhibited by rat high-density lipoprotein (HDL). The aim of this work was to investigate whether human lipoprotein lipase was also inhibited, the mechanism of inhibition of the rat enzyme by HDL, and the role of the various individual apolipoproteins. Both human and rat lipoprotein lipase from post-heparin plasma are inhibited by HDL. This inhibition is considerably decreased if the HDL is first made 'apolipoprotein poor' by removal of some transferable apolipoproteins. In contrast, both native and apolipoprotein poor HDL inhibit the hydrolysis of Intralipid by rat hepatic lipase. Apolipoproteins C and E, either free in solution or attached to lipid vesicles, inhibit the hydrolysis of activated Intralipid by rat lipoprotein lipase to a maximum of 85% and 50%, respectively. Apolipoprotein A attached to vesicles gives little inhibition. HDL apolipoprotein and apolipoprotein C compete with the substrate for binding to lipoprotein lipase with apolipoprotein C having a higher affinity for the enzyme than HDL apolipoprotein. The inhibition of lipoprotein lipase by HDL can be explained by the association of the constituent apolipoproteins, in particular apolipoprotein C, with the enzyme so that there is less enzyme available to act on substrate.     

Fluorescent lipoprotein probe

The fluorescent cholesterol analog cholesta-5,7,9(11)triene-3-β-ol was used to label high-density and low-density lipoproteins in vivo (rabbit) and in vitro (human). Rabbit feeding experiments demonstrated that in vivo both the esterified and nonesterified forms of the fluorophore were incorporated by these particles. Using in vitro labeling techniques, it was possible to selectively incorporate either the free form of the fluorophore or both the free and the esterified forms depending upon the presence or absence of serum esterases during incubation. Subsequent to labeling, the thermotropic behavior of the low- and high-density lipoproteins was evaluated using temperature-dependent fluorescence intensity measurements. Purified low-density lipoprotein samples (human and rabbit) containing both forms of the fluorophore were observed to undergo a thermotropic transition between 27 and 32°C. However, this transition was not observed in low-density lipoprotein samples containing only the nonesterified form of the probe nor was it observed in any of the high-density lipoprotein samples, even those containing both forms of the fluorophore. These results provide further evidence that the previously reported thermotropic transition in low-density lipoprotein is due to a reordering of the low-density lipoprotein cholesterol ester core (Deckelbaum, R.J., Shipley, G. G., Small, D. M., Lees, R. S., and George, P. K. (1975) Science190, 392–394; Sears, B., Deckelbaum, R. J., Janiak, M. J., Shipley, G. G., and Small, D. M. (1976) Biochemistry15, 4151–4157; Chana, G. S., Sheppard, R. J., Mills, G. L., and Grant, E. H. (1980) Phys. Med. Biol.25, 427–432).     

Plasma very-low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein oxidative modification induces procoagulant profiles in endogenous hypertriglyceridemia

This study was to investigate whether oxidatively modified lipoproteins were associated with changes of pro- and anticoagulant profiles in hypertriglyceridemic subjects. Plasma VLDL, LDL, and HDL were isolated with the one-step density gradient ultracentrifugation method. The oxidation of the lipoproteins was identified. Prothrombin time (PT) and activated partial thrombplastin time (APTT), tissue plasminogen activator and plasminogen activator inhibitor-1, and platelet aggregation rate were determined with a reaction system consisting of mixed fresh normal plasma, in endogenous hypertriglyceridemic (HTG) patients, in in vitro modified lipoproteins from a normolipidemic donor, and in experimental rats. The results indicated that oxVLDL, oxLDL, and oxHDL occurred in the plasma of HTG patients. Compared with the control group, PT and APTT, incubated with plasma VLDL, LDL, or HDL from HTG patients, respectively, were significantly reduced, while platelet maximal aggregation rates were significantly higher (P < 0.05-0.01). Similar procoagulant profiles were observed in in vitro modified lipoprotein components and in rats with intrinsic hypertriglyceridemia as well. These results support our previous finding that LDL, VLDL, and HDL were all oxidatively modified in vivo in the subjects with HTG, and suggest that procoagulation state may result from the abnormal plasma lipoprotein oxidative modification in vivo.     

Immunological relationship between bile lipoprotein complex and high density lipoprotein.

Apo bile lipoprotein complex was isolated from human gallbladder bile and an antiserum was prepared. Double immunodiffusion studies show that apo bile lipoprotein complex is present in human plasma and more precisely one of its proteic component is common to bile lipoprotein complex and HDL fraction. This proteic component is different from apo AI, AII, apo B, apo CI, CII, CIII, apo D, apo E and albumin.     

Triglyceride-rich lipoprotein cholesterol exceeds low-density lipoprotein cholesterol in hypertriglyceridemia patients.

The atherogenicity of triglyceride-rich lipoprotein has been revealed. This study was performed to explore the clinical importance of triglyceride-rich lipoprotein by measuring its cholesterol content and comparing it with other lipoprotein fractions. Blood samples were obtained from 103 patients whose fasting plasma triglyceride concentration exceeded 300 mg/dl. The cholesterol monitor using the technique of high-performance liquid chromatography was used for the measurement of their plasma cholesterol concentrations and the determination of cholesterol distribution among lipoprotein fractions. This monitor showed 4 peaks: large-triglyceride-rich lipoprotein, small-triglyceride-rich lipoprotein, low-density lipoprotein, and high-density lipoprotein. Total cholesterol increased with increasing triglyceride. The increment of total cholesterol was nearly equal to that of small-triglyceride-rich lipoprotein cholesterol. Small-triglyceride-rich lipoprotein cholesterol exceeded low-density lipoprotein cholesterol where plasma triglyceride concentration was over 500 mg/dl. In conclusion, triglyceride-rich lipoprotein may be clinically important for hypertriglyceridemic patients as a source of cholesteryl ester in arteriosclerotic plaques, and increased triglyceride-rich lipoprotein cholesterol may be used as a basis for hypertriglyceridemia atherogenicity. Our study suggests that hypertriglyceridemia should be treated to prevent arteriosclerotic disease.     

Achieving secondary prevention low-density lipoprotein particle concentration goals using lipoprotein cholesterol-based data

Background

Epidemiologic studies suggest that LDL particle concentration (LDL-P) may remain elevated at guideline recommended LDL cholesterol goals, representing a source of residual risk. We examined the following seven separate lipid parameters in achieving the LDL-P goal of <1000 nmol/L goal for very high risk secondary prevention: total cholesterol to HDL cholesterol ratio, TC/HDL, <3; a composite of ATP-III very high risk targets, LDL-C<70 mg/dL, non-HDL-C<100 mg/dL and TG<150 mg/dL; a composite of standard secondary risk targets, LDL-C<100, non-HDL-C<130, TG<150; LDL phenotype; HDL-C≥40; TG<150; and TG/HDL-C<3.

Methods

We measured ApoB, ApoAI, ultracentrifugation lipoprotein cholesterol and NMR lipoprotein particle concentration in 148 unselected primary and secondary prevention patients.

Results

TC/HDL-C<3 effectively discriminated subjects by LDL-P goal (F = 84.1, p<10⁻⁶). The ATP-III very high risk composite target (LDL-C<70, nonHDL-C<100, TG<150) was also effective (F = 42.8, p<10⁻⁵). However, the standard secondary prevention composite (LDL-C<100, non-HDL-C<130, TG<150) was also effective but yielded higher LDL-P than the very high risk composite (F = 42.0, p<10⁻⁵) with upper 95% confidence interval of LDL-P less than 1000 nmol/L. TG<150 and TG/HDL-C<3 cutpoints both significantly discriminated subjects but the LDL-P upper 95% confidence intervals fell above goal of 1000 nmol/L (F = 15.8, p = 0.0001 and F = 9.7, p = 0.002 respectively). LDL density phenotype neared significance (F = 2.85, p = 0.094) and the HDL-C cutpoint of 40 mg/dL did not discriminate (F = 0.53, p = 0.47) alone or add discriminatory power to ATP-III targets.

Conclusions

A simple composite of ATP-III very high risk lipoprotein cholesterol based treatment targets or TC/HDL-C ratio <3 most effectively identified subjects meeting the secondary prevention target level of LDL-P<1000 nmol/L, providing a potential alternative to advanced lipid testing in many clinical circumstances.     

Detection of new epitopes formed upon oxidation of low-density lipoprotein, lipoprotein (a) and very-low-density lipoprotein. Use of an antiserum against 4-hydroxynonenal-modified low-density lipoprotein.

4-Hydroxynonenal (HNE) is a major aldehydic propagation product formed during peroxidation of unsaturated fatty acids. The aldehyde was used to modify freshly prepared human low-density lipoprotein (LDL). A polyclonal antiserum was raised in the rabbit and absorbed with freshly prepared LDL. The antiserum did not react with human LDL, but reacted with CuCl2-oxidized LDL and in a dose-dependent manner with LDL, modified with 1, 2 and 3 mM-HNE, in the double-diffusion analysis. LDL treated with 4 mM of hexanal or hepta-2,4-dienal or 4-hydroxyhexenal or malonaldehyde (4 or 20 mM) did not react with the antiserum. However, LDL modified with 4 mM-4-hydroxyoctenal showed a very weak reaction. Lipoprotein (a) and very-low-density lipoprotein were revealed for the first time to undergo oxidative modification initiated by CuCl2. This was evidenced by the generation of lipid hydroperoxides and thiobarbituric acid-reactive substances, as well as by a marked increase in the electrophoretic mobility. After oxidation these two lipoproteins also reacted positively with the antiserum against HNE-modified LDL.     

Heterogeneity of lipoprotein B

Combined very low and low density lipoproteins were derived from human plasma by polyanion precipitation and the low density lipoprotein fraction (density 1.027–1.050 g/ml) was isolated by sequential ultracentrifugation. When this fraction was applied to Sepharose column chromatography, three lipoproteins were eluted. The first and third peaks were minor components while the second peak represented the bulk of LDL. Further chromatographic and electrophoretic studies indicated that the component representing the second peak was heterogeneous. This component was subsequently delipidated at pH 4 in a quaternary biphasic solvent system. The apoproteins remained soluble after delipidation and were treated with various deaggregating agents. On column isoelectric focusing in the presence of 4 M urea the apoproteins banded as broad overlapping peaks between pH 3 and 7. When hexanol was added to the system, distinct apoprotein subfractions were resolved.     

Musca domestica larval lipoprotein

A larval specific high-density lipoprotein (HDL) has been isolated from Musca domestica hemolymph by a combination of density gradient and glycerol gradient ultracentrifugations. The larval lipoprotein has a density of 1.134 g/ml and is formed by at least four apoproteins with molecular weights equal to 26,000, 23,000, 21,000, and 20,000. This lipoprotein contains large amounts of hydrocarbons and phospholipids and minor amounts of diacylglycerols and cholesterol. The larval lipoprotein is completely distinct from lipophorin in regard to apoprotein composition, lipid moiety, physiological pattern, and immunological reactions. Larval lipoprotein is accumulated until the end of the feeding period. During the pupal molt this protein is utilized and is no longer detected after 2 days of pupal stadium. The results obtained imply a possible role of this protein in the puparia and/or pupal cuticle formation. Judging from the properties shown, the Musca domestica larval lipoprotein is a completely new type of insect lipoprotein.