Fenofibrate therapy and cardiovascular protection in diabetes: recommendations after FIELD

PURPOSE OF REVIEW: The aim of the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study was to provide valuable information on the ability of fibrates to reduce cardiovascular risk in type 2 diabetes. The purpose of this review is to analyse results from FIELD and to see whether they may lead to modify the recommendations for treatment of diabetic dyslipidemia. RECENT FINDINGS: In FIELD, fenofibrate therapy was associated with a nonsignificant 11% reduction in the primary endpoint (coronary heart disease death, nonfatal myocardial infarction), corresponding to a significant 24% reduction in nonfatal myocardial infarction (P = 0.010) and a nonsignificant 19% increase in CHD mortality. Fenofibrate reduced CHD events only in patients in primary prevention, but not in secondary prevention. Fenofibrate treatment was associated with less albuminuria progression and less retinopathy needing laser treatment. SUMMARY: FIELD's results are somewhat disappointing. The relatively low cardiovascular risk population from FIELD and the 'pollution' by statin therapy may not totally explain the weak results of fenofibrate in the reduction in CHD events. The significant increase in plasma homocysteine observed with fenofibrate could partly explain not only the higher number of venous thrombotic events, but also the poor effect of fenofibrate in reducing clinical outcomes, more particularly in patients with previous cardiovascular disease.     

HDL metabolism in context: looking on the bright side

PURPOSE OF REVIEW: To review new data concerning HDL metabolism and cardiovascular disease, the concept of HDL 'functionality', and HDL kinetics in the metabolic syndrome. RECENT FINDINGS: HDL-apoA-I and apoA-II may be better predictors of cardiovascular disease than HDL-cholesterol. Cholesteryl ester transfer protein inhibition with torcetrapib does not benefit cardiovascular disease; whether this is related to 'congestion' of HDL transport or a specific off-target vasopressor effect remains unclear. Accelerated catabolism of HDL particles in metabolic syndrome could be due to increased hepatic secretion of apoB and apoC-III, hepatic steatosis, and low plasma adiponectin. The role of serum amyloid A and homocysteine is uncertain. In metabolic syndrome, therapies that could favourably alter HDL transport include weight loss, fish oils, higher dose statins, and fibrates; 'balancing feedback' may offset reduced catabolism of HDL, fenofibrate being the only agent hitherto shown to increase apoA-I production. SUMMARY: Elevating HDL-apoA-I and apoA-II may be a more important therapeutic objective than increased HDL-cholesterol. Recent studies underscore the potential value of studying HDL functionality, particularly in the metabolic syndrome. Reverse cholesterol transport can only be reliably probed at present by studying the kinetics of HDL particles or apolipoproteins; new methods are needed for investigating cellular and whole body cholesterol turnover. In metabolic syndrome, HDL-raising therapies have differential impact on HDL kinetics, the optimal endpoint being to increase transport and concentration with unchanged or accelerated catabolism.     

Fenofibrate reverses the decline in HDL cholesterol in mice overexpressing human phospholipid transfer protein

In humans, fibrates are used to treat dyslipidemia, because these drugs lower plasma triglycerides and raise HDL cholesterol. Treatment with fibrates lowers plasma phospholipid transfer protein (PLTP) activity in humans, but increases PLTP activity in mice, without a consistent effect on HDL-cholesterol concentration. Earlier, we found that PLTP overexpression in transgenic mice results in decreased plasma HDL levels and increased diet-induced atherosclerosis. So it seems that the interplay between fibrates, PLTP and HDL is different in mice and man, which may be important for atherosclerosis development. In the present study, we measured the effects of fibrates on PLTP expression in cultured human hepatocytes and effects of fibrate treatment on human PLTP expression, plasma PLTP activity and HDL levels in human PLTP transgenic mice. Fibrate treatment did not influence PLTP mRNA levels in human hepatocytes. Hepatic human PLTP mRNA levels and PLTP activity were both moderately elevated by fenofibrate treatment in human PLTP transgenic mice. In wild-type mice, however, feeding fenofibrate resulted in a strong induction of PLTP mRNA in the liver and a more than 4-fold increase of plasma PLTP activity. Plasma triglycerides were reduced in all mice by 48% or more by fenofibrate treatment. HDL-cholesterol concentrations were substantially increased by fenofibrate in PLTP overexpressing mice (+72%), but unaffected in wild-type mice. We conclude that fenofibrate treatment reverses the HDL-lowering effect of PLTP overexpression in human PLTP transgenic mice.     

ApoA-II maintains HDL levels in part by inhibition of hepatic lipase. Studies In apoA-II and hepatic lipase double knockout mice.

High density lipoprotein (HDL) cholesterol levels are inversely related to the risk of developing coronary heart disease. Apolipoprotein (apo) A-II is the second most abundant HDL apolipoprotein and apoA-II knockout mice show a 70% reduction in HDL cholesterol levels. There is also evidence, using human apoA-II transgenic mice, that apoA-II can prevent hepatic lipase-mediated HDL triglyceride hydrolysis and reduction in HDL size. These observations suggest the hypothesis that apoA-II maintains HDL levels, at least in part, by inhibiting hepatic lipase. To evaluate this, apoA-II knockout mice were crossbred with hepatic lipase knockout mice. Compared to apoA-II-deficient mice, in double knockout mice there were increased HDL cholesterol levels (57% in males and 60% in females), increased HDL size, and decreased HDL cholesteryl ester fractional catabolic rate. In vitro incubation studies of plasma from apoA-II knockout mice, which contains largely apoA-I HDL particles, showed active lipolysis of HDL triglyceride, whereas similar studies of plasma from apoA-I knockout mice, which contains largely apoA-II particles, did not. In summary, these results strongly suggest that apoA-II is a physiological inhibitor of hepatic lipase and that this is at least part of the mechanism whereby apoA-II maintains HDL cholesterol levels.     

A polymorphism affecting apolipoprotein A-II translational efficiency determines high density lipoprotein size and composition

High density lipoproteins (HDL) are heterogeneous particles consisting of about equal amounts of lipid and protein that are thought to mediate the transport of cholesterol from peripheral tissues to liver. We show that a previously identified polymorphism affecting HDL electrophoretic mobility in mice is due to a monogenic variation controlling HDL size and apolipoprotein composition. Thus, the HDL particles of various inbred strains of mice exhibit a striking difference in the ratio fo the two major apolipoproteins of HDL, apoA-I and apoA-II. HDL particles in all strains examined contain an average of about five apoA-I molecules; however, whereas the strains with small HDL contain two to three apoA-II molecules per particle, the strains with large HDL contain about five apoA-II molecules per particle. This increase in the protein content of the large HDL is also accompanied by increased lipid content. The HDL size polymorphism and apoA-II levels cosegregate with the apoA-II structural gene on mouse chromosome 1, indicating that a mutation of the apoA-II gene locus is responsible. The rates of synthesis of apoA-II are increased in the strains with large HDL and high apoA-II levels as compared to the strains with small HDL and low apoA-II levels. On the other hand, the fractional catabolic rates of both apoA-I and apoA-II among the strains are very similar, confirming that apoA-II concentrations are controlled at the level of synthesis. Despite the difference in rates of apoA-II synthesis between strains, the apoA-II mRNA levels in the strains are not discernibly different, suggesting that a mutation of the apoA-II structural gene controls apoA-II translational efficiency. This was confirmed by translating apoA-II mRNA in vitro using a rabbit reticulocyte lysate system. Sequencing of apoA-II cDNA from the strains revealed a number of nucleotide substitutions, which may affect translational efficiency. We conclude that the assembly of apoA-II into HDL does not have a set stoichiometry but, rather, is controlled by the production of apoA-II. As apoA-II levels increase, the HDL particles become larger and acquire more lipid, but apoA-I content per particle remains unchanged. These studies with mice provide a model for the metabolic relationships between apoA-I, apoA-II, and HDL lipid in humans.     

Role of apoA-II in lipid metabolism and atherosclerosis: advances in the study of an enigmatic protein.

Our understanding of apolipoprotein A-II (apoA-II) physiology is much more limited than that of apoA-I. However, important and rather surprising advances have been produced, mainly through analysis of genetically modified mice. These results reveal a positive association of apoA-II with FFA and VLDL triglyceride plasma concentrations; however, whether this is due to increased VLDL synthesis or to decreased VLDL catabolism remains a matter of controversy. As apoA-II-deficient mice present a phenotype of insulin hypersensitivity, a function of apoA-II in regulating FFA metabolism seems likely. Studies of human beings have shown the apoA-II locus to be a determinant of FFA plasma levels, and several genome-wide searches of different populations with type 2 diabetes have found linkage to an apoA-II intragenic marker, making apoA-II an attractive candidate gene for this disease. The increased concentration of apoB-containing lipoproteins present in apoA-II transgenic mice explains, in part, why these animals present increased atherosclerosis susceptibility. In addition, apoA-II transgenic mice also present impairment of two major HDL antiatherogenic functions: reverse cholesterol transport and protection of LDL oxidative modification. The apoA-II locus has also been suggested as an important genetic determinant of HDL cholesterol concentration, even though there is a major species-specific difference between the effects of mouse and human apoA-II. As antagonizing apoA-I antiatherogenic actions can hardly be considered the apoA-II function in HDL, this remains a topic for future investigations. We suggest that the existence of apoA-II or apoA-I in HDL could be an important signal for specific interaction with HDL receptors such as cubilin or heat shock protein 60.     

Spur cells in patients with alcoholic liver cirrhosis are associated with reduced plasma levels of apoA-II, HDL3, and LDL

The precise nature and origin(s) of the abnormalities in lipoprotein and apolipoprotein profile associated with severe hepatic dysfunction and the presence of spur cells remain poorly defined. To shed light on this question, we have analyzed the plasma lipoprotein and apolipoprotein profiles in five patients with alcoholic cirrhosis and spur cells, and compared them with those of a group with similar hepatocellular dysfunction, but lacking spur cells, and with that of a control group. Lipoproteins were subfractionated by density gradient ultracentrifugation and their physicochemical properties were determined; apolipoprotein A-I, A-II, and B contents in plasma and the respective subfractions were quantitated by radial immunodiffusion, while the complement of low molecular weight apolipoproteins in each subfraction was analyzed by isoelectric focusing and electrophoresis in alkaline-urea polyacrylamide gels. Spur cell plasma was distinguished by reduced levels of apoA-II and elevated ratios of apoA-I/apoA-II (approximately 13:1 as compared to 3.3-3.9:1 in the other two groups), and by reduced concentrations of HDL3. Gradient fractionation showed the apoA-II content of HDL3 to be dramatically and significantly diminished in spur cell plasma; in addition, apoA-II content was reduced relative to apoA-I in this subclass (4.7:1 as compared to 1:1 in cirrhotics lacking spur cells and 1.9:1 in controls). Spur cell HDL2 was similarly deficient in apoA-II, with elevated ratios of apoA-I:apoA-II (9.8:1 in comparison with 1.9-2.5:1 in the two other groups). Nonetheless, high HDL2 concentrations were seen in both series of cirrhotic patients, irrespective of red cell morphology. Spur cell HDL2 thus appears to consist primarily of particles possessing only apoA-I, with a minor population containing both apoA-I and apoA-II. The free cholesterol content of all lipoprotein subfractions from spur cell plasma was increased, as indeed was the molar ratio of free cholesterol to phospholipid, in comparison with that of corresponding fractions from alcoholic cirrhotics lacking spur cells and of control subjects. LDL levels were reduced in spur cell plasma, thereby distinguishing this group from the cirrhotics without spur cells who displayed elevated LDL levels. Markedly reduced plasma levels of apoA-II, HDL3, and LDL appear characteristic of alcoholic cirrhotics presenting with spur cells. Our findings suggest that apoA-II may be essential to the normal function and metabolism of HDL, one aspect of which may be the transport of free cholesterol and thereby the direct or indirect maintenance of red cell morphology.     

Induction of the phospholipid transfer protein gene accounts for the high density lipoprotein enlargement in mice treated with fenofibrate

Fibrate treatment in mice is known to modulate high density lipoprotein (HDL) metabolism by regulating apolipoprotein (apo)AI and apoAII gene expression. In addition to alterations in plasma HDL levels, fibrates induce the emergence of large, cholesteryl ester-rich HDL in treated transgenic mice expressing human apoAI (HuAITg). The mechanisms of these changes may not be restricted to the modulation of apolipoprotein gene expression, and the aim of the present study was to determine whether the expression of factors known to affect HDL metabolism (i.e. phospholipid transfer protein (PLTP), lecithin:cholesterol acyltransferase, and hepatic lipase) are modified in fenofibrate-treated mice. Significant rises in plasma PLTP activity were observed after 2 weeks of fenofibrate treatment in both wild-type and HuAITg mice. Simultaneously, hepatic PLTP mRNA levels increased in a dose-dependent fashion. In contrast to PLTP, lecithin:cholesterol acyltransferase mRNA levels in HuAITg mice were not significantly modified by fenofibrate despite a significant decrease in plasma cholesterol esterification activity. Fenofibrate did not induce any change in hepatic lipase activity. Fenofibrate significantly increased HDL size, an effect that was more pronounced in HuAITg mice than in wild-type mice. This effect in wild-type mice was completely abolished in PLTP-deficient mice. Finally, fenofibrate treatment did not influence PLTP activity or hepatic mRNA in peroxisome proliferator-activated receptor-alpha-deficient mice. It is concluded that 1) fenofibrate treatment increases plasma phospholipid transfer activity as the result of up-regulation of PLTP gene expression through a peroxisome proliferator-activated receptor-alpha-dependent mechanism, and 2) increased plasma PLTP levels account for the marked enlargement of HDL in fenofibrate-treated mice.     

In vivo interactions of apoA-II, apoA-I, and hepatic lipase contributing to HDL structure and antiatherogenic functions

Studies with mice have revealed that increased expression of apolipoprotein A-II (apoA-II) results in elevations in high density lipoprotein (HDL), the formation of larger HDL, and the development of early atherosclerosis. We now show that the increased size of HDL results in part from an inhibition of the ability of hepatic lipase (HL) to hydrolyze phospholipids and triglycerides in the HDL and that the ratio of apoA-I to apoA-II determines HDL functional and antiatherogenic properties. HDL from apoA-II transgenic mice was relatively resistant to the action of HL in vitro. To test whether HL and apoA-II influence HDL size independently, combined apoA-II transgenic/HL knockout (HLko) mice were examined. These mice had HDL similar in size to apoA-II transgenic mice and HLko mice, suggesting that they do not increase HDL side by independent mechanisms. Overexpression of apoA-I from a transgene reversed many of the effects of apoA-II overexpression, including the ability of HDL to serve as a substrate for HL. Combined apoA-I/apoA-II transgenic mice exhibited significantly less atherosclerotic lesion formation than did apoA-II transgenic mice. These results were paralleled by the effects of the transgenes on the ability of HDL to protect against the proinflammatory effects of oxidized low density lipoprotein (LDL). Whereas nontransgenic HDL protected against oxidized LDL induction of adhesion molecules in endothelial cells, HDL from apoA-II transgenic mice was proinflammatory. HDL from combined apoA-I/apoA-II transgenic mice was equally as protective as HDL from nontransgenic mice. Our data suggest that as the ratio of apoA-II to apoA-I is increased, the HDL become larger because of inhibition of HL, and lose their antiatherogenic properties.     

The structure of apolipoprotein A-II in discoidal high density lipoproteins

It is well accepted that high levels of high density lipoproteins (HDL) reduce the risk of atherosclerosis in humans. Apolipoprotein A-I (apoA-I) and apoA-II are the first and second most common protein constituents of HDL. Unlike apoA-I, detailed structural models for apoA-II in HDL are not available. Here, we present a structural model of apoA-II in reconstituted HDL (rHDL) based on two well established experimental approaches: chemical cross-linking/mass spectrometry (MS) and internal reflection infrared spectroscopy. Homogeneous apoA-II rHDL were reacted with a cross-linking agent to link proximal lysine residues. Upon tryptic digestion, cross-linked peptides were identified by electrospray mass spectrometry. 14 cross-links were identified and confirmed by tandem mass spectrometry (MS/MS). Infrared spectroscopy indicated a beltlike molecular arrangement for apoA-II in which the protein helices wrap around the lipid bilayer rHDL disc. The cross-links were then evaluated on three potential belt arrangements. The data clearly refute a parallel model but support two antiparallel models, especially a "double hairpin" form. These models form the basis for understanding apoA-II structure in more complex HDL particles.     

ApoA-I deficiency in mice is associated with redistribution of apoA-II and aggravated AApoAII amyloidosis

Apolipoprotein A-II (apoA-II) is the second major apolipoprotein following apolipoprotein A-I (apoA-I) in HDL. ApoA-II has multiple physiological functions and can form senile amyloid fibrils (AApoAII) in mice. Most circulating apoA-II is present in lipoprotein A-I/A-II. To study the influence of apoA-I on apoA-II and AApoAII amyloidosis, apoA-I-deficient (C57BL/6J.Apoa1^−/−) mice were used. Apoa1^−/− mice showed the expected significant reduction in total cholesterol (TC), HDL cholesterol (HDL-C), and triglyceride (TG) plasma levels. Unexpectedly, we found that apoA-I deficiency led to redistribution of apoA-II in HDL and an age-related increase in apoA-II levels, accompanied by larger HDL particle size and an age-related increase in TC, HDL-C, and TG. Aggravated AApoAII amyloidosis was induced in Apoa1^−/− mice systemically, especially in the heart. These results indicate that apoA-I plays key roles in maintaining apoA-II distribution and HDL particle size. Furthermore, apoA-II redistribution may be the main reason for aggravated AApoAII amyloidosis in Apoa1^−/− mice. These results may shed new light on the relationship between apoA-I and apoA-II as well as provide new information concerning amyloidosis mechanism and therapy.     

ApoA-II modulates the association of HDL with class B scavenger receptors SR-BI and CD36

The class B scavenger receptors SR-BI and CD36 exhibit a broad ligand binding specificity. SR-BI is well characterized as a HDL receptor that mediates selective cholesteryl ester uptake from HDL. CD36, a receptor for oxidized LDL, also binds HDL and mediates selective cholesteryl ester uptake, although much less efficiently than SR-BI. Apolipoprotein A-II (apoA-II), the second most abundant HDL protein, is considered to be proatherogenic, but the underlying mechanisms are unclear. We previously showed that apoA-II modulates SR-BI-dependent binding and selective uptake of cholesteryl ester from reconstituted HDL. To investigate the effect of apoA-II in naturally occurring HDL on these processes, we compared HDL without apoA-II (from apoA-II null mice) with HDLs containing differing amounts of apoA-II (from C57BL/6 mice and transgenic mice expressing a mouse apoA-II transgene). The level of apoA-II in HDL was inversely correlated with HDL binding and selective cholesteryl ester uptake by both scavenger receptors, particularly CD36. Interestingly, for HDL lacking apoA-II, the efficiency with which CD36 mediated selective uptake reached a level similar to that of SR-BI. These results demonstrate that apoA-II exerts a marked effect on HDL binding and selective lipid uptake by the class B scavenger receptors and establishes a potentially important relationship between apoA-II and CD36.     

Effect of rosiglitazone on HDL metabolism in subjects with metabolic syndrome and low HDL

Treatment with the peroxisome proliferator-activated receptor γ agonist rosiglitazone has been reported to increase HDL-cholesterol (HDL-C) levels, although the mechanism responsible for this is unknown. We sought to determine the effect of rosiglitazone on HDL apolipoprotein A-I (apoA-I) and apoA-II metabolism in subjects with metabolic syndrome and low HDL-C. Subjects were treated with placebo followed by rosiglitazone (8 mg) once daily. At the end of each 8 week treatment, subjects (n = 15) underwent a kinetic study to measure apoA-I and apoA-II production rate (PR) and fractional catabolic rate. Rosiglitazone significantly reduced fasting insulin and high-sensitivity C-reactive protein (hsCRP) and increased apoA-II levels. Mean apoA-I and HDL-C levels were unchanged following rosiglitazone treatment, although there was considerable individual variability in the HDL-C response. Rosiglitazone had no effect on apoA-I metabolism, whereas the apoA-II PR was increased by 23%. The change in HDL-C in response to rosiglitazone was significantly correlated with the change in apoA-II concentration but not to changes in apoA-I, measures of glucose homeostasis, or hsCRP. Treatment with rosiglitazone significantly increased apoA-II production in subjects with metabolic syndrome and low HDL-C but had no effect on apoA-I metabolism. The change in HDL-C in response to rosiglitazone treatment was unrelated to effects on apoA-I, instead being related to the change in the metabolism of apoA-II.     

Clustering by plasma lipoprotein profile reveals two distinct subgroups with positive lipid response to fenofibrate therapy

Fibrates lower triglycerides and raise HDL cholesterol in dyslipidemic patients, but show heterogeneous treatment response. We used k-means clustering to identify three representative NMR lipoprotein profiles for 775 subjects from the GOLDN population, and study the response to fenofibrate in corresponding subgroups. The subjects in each subgroup showed differences in conventional lipid characteristics and in presence/absence of cardiovascular risk factors at baseline; there were subgroups with a low, medium and high degree of dyslipidemia. Modeling analysis suggests that the difference between the subgroups with low and medium dyslipidemia is influenced mainly by hepatic uptake dysfunction, while the difference between subgroups with medium and high dyslipidemia is influenced mainly by extrahepatic lipolysis disfunction. The medium and high dyslipidemia subgroups showed a positive, yet distinct lipid response to fenofibrate treatment. When comparing our subgroups to known subgrouping methods, we identified an additional 33% of the population with favorable lipid response to fenofibrate compared to a standard baseline triglyceride cutoff method. Compared to a standard HDL cholesterol cutoff method, the addition was 18%. In conclusion, by using constructing subgroups based on representative lipoprotein profiles, we have identified two subgroups of subjects with positive lipid response to fenofibrate therapy and with different underlying disturbances in lipoprotein metabolism. The total subgroup with positive lipid response to fenofibrate is larger than subgroups identified with baseline triglyceride and HDL cholesterol cutoffs.     

Isoproteins of human apolipoprotein A-II: isolation and characterization

In human serum, polymorphism of apoA-II predominantly in HDL3 could be demonstrated. HDL3-apoA-II was composed of four isoproteins, each with a molecular weight of 8600 (reduced form) and identical immunological properties. The isoproteins are designated apoA-II-1 (pI 5.16), apoA-II-2 (pI 4.89) corresponding to the already known apoA-II monomer band, apoA-II-3 (pI 4.58), and apoA-II-4 (pI 4.31). The amino acid compositions of the A-II isoproteins were virtually identical with the published data for apoA-II. Treatment with acid phosphatase, alkaline phosphatase, or neuraminidase before electrophoresis did not alter the apoA-II pattern. The apoA-II isoprotein pattern was studied in ten male and ten female normolipidemic volunteers, in two patients with Tangier disease, and in three patients with abetalipoproteinemia. The isoelectric focusing patterns of apoA-II appeared virtually identical in all subjects. However, in Tangier disease, due to the low apo-A-II concentration, only apoA-II-1 and apoA-II-2 were detectable, and in abetalipoproteinemia a different relative distribution pattern of the individual isoforms was found as compared to normal HDL3. Our studies indicate that apoA-II, similar to apoA-I, exists in several isoforms. The relationship of these isoforms to each other is at present unclear. They may originate from relatively basic isoproteins that are modified in charge by post-translational processes such as proteolytic cleavage, sequential deamidation, or other mechanisms.     

Apolipoprotein A-II is catabolized in the kidney as a function of its plasma concentration

We investigated in vivo catabolism of apolipoprotein A-II (apo A-II), a major determinant of plasma HDL levels. Like apoA-I, murine apoA-II (mapoA-II) and human apoA-II (hapoA-II) were reabsorbed in the first segment of kidney proximal tubules of control and hapoA-II-transgenic mice, respectively. ApoA-II colocalized in brush border membranes with cubilin and megalin (the apoA-I receptor and coreceptor, respectively), with mapoA-I in intracellular vesicles of tubular epithelial cells, and was targeted to lysosomes, suggestive of degradation. By use of three transgenic lines with plasma hapoA-II concentrations ranging from normal to three times higher, we established an association between plasma concentration and renal catabolism of hapoA-II. HapoA-II was rapidly internalized in yolk sac epithelial cells expressing high levels of cubilin and megalin, colocalized with cubilin and megalin on the cell surface, and effectively competed with apoA-I for uptake, which was inhibitable by anti-cubilin antibodies. Kidney cortical cells that only express megalin internalized LDL but not apoA-II, apoA-I, or HDL, suggesting that megalin is not an apoA-II receptor. We show that apoA-II is efficiently reabsorbed in kidney proximal tubules in relation to its plasma concentration.     

Formation of high density lipoproteins containing both apolipoprotein A-I and A-II in the rabbit

Human plasma HDLs are classified on the basis of apolipoprotein composition into those that contain apolipoprotein A-I (apoA-I) without apoA-II [(A-I)HDL] and those containing apoA-I and apoA-II [(A-I/A-II)HDL]. ApoA-I enters the plasma as a component of discoidal particles, which are remodeled into spherical (A-I)HDL by LCAT. ApoA-II is secreted into the plasma either in the lipid-free form or as a component of discoidal high density lipoproteins containing apoA-II without apoA-I [(A-II)HDL]. As discoidal (A-II)HDL are poor substrates for LCAT, they are not converted into spherical (A-II)HDL. This study investigates the fate of apoA-II when it enters the plasma. Lipid-free apoA-II and apoA-II-containing discoidal reconstituted HDL [(A-II)rHDL] were injected intravenously into New Zealand White rabbits, a species that is deficient in apoA-II. In both cases, the apoA-II was rapidly and quantitatively incorporated into spherical (A-I)HDL to form spherical (A-I/A-II)HDL. These particles were comparable in size and composition to the (A-I/A-II)HDL in human plasma. Injection of lipid-free apoA-II and discoidal (A-II)rHDL was also accompanied by triglyceride enrichment of the endogenous (A-I)HDL and VLDL as well as the newly formed (A-I/A-II)HDL. We conclude that, irrespective of the form in which apoA-II enters the plasma, it is rapidly incorporated into spherical HDLs that also contain apoA-I to form (A-I/A-II)HDL.     

Effects of peroxisome proliferator-activated receptor alpha/delta agonists on HDL-cholesterol in vervet monkeys

The objective of this study was to demonstrate the efficacy of a novel peroxisome proliferator-activated receptor (PPAR) agonist and known PPARalpha and PPARdelta agonists to increase HDL-cholesterol (HDL-C) in the St. Kitts vervet, a nonhuman primate model of atherosclerosis. Four groups (n = 6) were studied and each group was assigned one of the following "treatments": a) vehicle only (vehicle); b) the PPARdelta selective agonist GW501516 (GW); c) the PPARalpha/delta agonist T913659 (T659); and d) the PPARalpha agonist TriCor (fenofibrate). No statistically significant changes were seen in body weight, total plasma cholesterol, plasma triglycerides, VLDL-C, LDL-C, or apolipoprotein B (apoB) concentrations. Each of the PPARalpha and PPARdelta agonists investigated in this study increased plasma HDL-C, apoA-I, and apoA-II concentrations and increased HDL particle size in St. Kitts vervets. The maximum percentage increase in HDL-C from baseline for each group was as follows: vehicle, 5%; GW, 43%; T659, 43%; and fenofibrate, 20%. Treatment with GW and T659 resulted in an increase in medium-sized HDL particles, whereas fenofibrate showed increases in large HDL particles. These data provide additional evidence that PPARalpha and PPARdelta agonists (both mixed and selective) have beneficial effects on HDL-C in these experimental primates.     

Combination of dietary phytosterols plus niacin or fenofibrate: effects on lipid profile and atherosclerosis in apo E-KO mice

Patients with mixed dyslipidemias (increased LDL cholesterol and triglyceride as well as low HDL cholesterol levels) benefit from a combination of lipid-modifying drugs such as statins, niacin, fibrates and ezetemibe. However, safety, tolerability and cost are a concern in drug combination therapy. Dietary phytosterols reduce LDL cholesterol, and niacin or fenofibrate primarily reduces triglyceride and increases HDL-cholesterol levels. Thus, we hypothesized that a combination of phytosterols with niacin or fenofibrate will synergistically impact lipoprotein profile and atherogenesis in apo E-KO mice. Phytosterols alone significantly reduced plasma total cholesterol levels (14.1 vs. 16.9 mmol/L, P < .05) and the extent of atherosclerosis (0.42 vs. 0.15 mm(2), P < .05). The addition of fenofibrate to phytosterols increased plasma total cholesterol levels by >50% (14.1 vs. 21.6 mmol/L, P < .05) and decreased HDL-cholesterol concentrations by 50% (0.8 vs. 0.4 mmol/L). These changes were accompanied by slight reductions in the extent of atherosclerosis (0.42 vs. 0.34 mm(2), P > 0.05) as compared to controls, suggesting other potential anti-atherogenic effects of fenofibrate. Unlike fenofibrate, niacin caused an increase of 150% (P < .05) in HDL-cholesterol concentrations and a decrease of 22% (P < .05) in total cholesterol levels which were associated with significant reductions (65%, P < .05) in atherosclerotic lesion size as compared to controls. Neither the addition of niacin nor of fenofibrate reduced plasma triglyceride levels. In conclusion, the addition of niacin to phytosterols synergistically increases HDL-cholesterol levels, while a combination of phytosterols and fenofibrate results in no synergistic effects in apo E-KO mice. Further studies in other animal models are needed to establish synergetic effects between these lipid-modifying dietary and pharmacological agents.     

The molecular structure of apolipoprotein A-II modulates the capacity of HDL to promote cell cholesterol efflux

The influence of apolipoprotein A-II (apoA-II) molecular structure on the capacity of high density lipoproteins (HDL) to promote cellular cholesterol efflux was investigated in cultured mouse peritoneal macrophages (MPM). Conversion by reduction and carboxamidomethylation of the naturally occurring dimeric apoA-II to its monomeric form in both native or reconstituted HDL did not change apolipoprotein secondary structure and lipoprotein size/composition. All particles containing monomeric apoA-II, i.e., native HDL₃ or reconstituted HDL with or without apoA-I, showed a higher ability to promote cholesterol efflux originating from plasma membrane and intracellular stores, compared to particles containing dimeric apoA-II. These findings indicate that apolipoprotein molecular structure is a major determinant of HDL capacity to promote cholesterol efflux from cells.