Search

Probucol is a powerful inhibitor of atherosclerosis in a number of animal models. However, it is unknown whether this is due to the strong antioxidant protection of low density lipoprotein (LDL), to antioxidant effects in the artery wall, or to cellular effects not shared by other antioxidants. To investigate whether murine models are suitable to study the antiatherogenic mechanisms of probucol, three experiments following different protocols were carried out in 135 male and female LDL receptor-deficient (LDLR-/-) mice. Treatment groups received a high (0.5%) or low (0.025%) dose of probucol, or low-dose probucol plus a high dose (0.1%) of vitamin E for periods ranging from 6 to 26 weeks. In all experiments, probucol strongly protected LDL against ex vivo oxidation (lag times exceeding 1400 min in 0.5% probucol-treated mice). Treatment with 0.5% probucol significantly lowered both HDL-cholesterol and plasma apolipoprotein (apo)A-I concentrations. In all three experiments, treatment with 0.5% probucol consistently increased the size of lesions in the aortic origin, from 1.3-fold (n.s.) to 2.9-fold (P < 0.05) in female mice and from 3.6- to 3.7-fold in males (P < 0.001). Even treatment with 0.025% probucol increased atherosclerosis 1.6-fold in male mice (P < 0.01). Addition of the high dose of vitamin E did not attenuate the pro-atherogenic effect of 0.025% probucol. In conclusion, probucol not only failed to decrease but actively increased atherogenesis in LDLR-/- mice in a dose-dependent manner, even though it provided a very strong antioxidant protection of LDL. This suggests that the reduction of atherosclerosis observed in other animal models is due to intracellular effects of probucol not found in mice, to differences in the metabolism of probucol, and/or to an overriding atherogenic effect of the decrease in HDL in murine models.

This year marks the 100th anniversary of the publication of Anitschkow's classic paper proposing the central role of hypercholesterolemia in atherogenesis. We at the Journal of Lipid Research take this occasion to acknowledge the debt we all owe to Anitschkow and his colleagues for getting us on the right track. As discussed below in detail, his contributions were insightful and went well beyond simply pinpointing hypercholesterolemia as a major etiologic factor. Anitschkow's work led him to define most of the key elements in the initiation and evolution of lesions in animal models of atherogenesis.

The accumulated evidence that oxidative modification of LDL plays an important role in the pathogenesis of atherosclerosis in animal models is very strong. The negative results in recent clinical studies have caused many to conclude that LDL oxidation may not be relevant in the human disease. Yet many of the lines of evidence that support the hypothesis have been demonstrated to apply also in humans. In this review, we briefly summarize the lines of evidence on which the hypothesis rests, its strengths, and its weaknesses.

The first four reviews in this series (Steinberg, D. 2004. J. Lipid Res. 45: 1583-1593; Steinberg, D. 2005. J. Lipid Res. 46: 179-190; Steinberg, D. 2005. J. Lipid Res. 46: 2037-2051; Steinberg, D. 2006. J. Lipid Res. 47: 1-14) traced the gradual accumulation of evidence, evidence of several different kinds, supporting the lipid hypothesis. They tracked the history from Anitschkow's 1913 classic work on the cholesterol-fed rabbit model to the breakthrough 1984 Coronary Primary Prevention Trial, the first large, randomized, double-blind primary intervention trial showing that decreasing blood cholesterol (using cholestyramine) significantly reduces coronary heart disease events. At that point, for the first time, decreasing blood cholesterol levels became an official national public health goal. Still, only a small fraction of patients at high risk were getting appropriate cholesterol-lowering treatment, and a number of important clinical questions remained unanswered. This final review in the series traces the early studies that led to the discovery of the statins and briefly reviews the now familiar large-scale clinical trials demonstrating their safety and their remarkable effectiveness in reducing coronary heart disease morbidity and mortality.

As of the early 1980s, despite the wealth of evidence from experimental animal models, the extensive epidemiologic evidence, the powerful genetic evidence, and the strongly suggestive clinical intervention trial results, most clinicians still remained unpersuaded regarding the relevance of the lipid hypothesis. What was needed was a well-designed, large-scale, long-term, double-blind study demonstrating a statistically significant impact of treatment on coronary heart disease events. The National Institutes of Health (NIH) had laid the groundwork for such a study as early as 1970, but the study was not completed and the results published until 1984. This study, the Coronary Primary Prevention Trial, showed that treatment with a bile acid binding resin reduced major coronary events in hypercholesterolemic men by 19%, with a P value of 0.05. The NIH followed this up with a national Consensus Development Conference on Lowering Blood Cholesterol to Prevent Heart Disease. For the first time, the NIH now went on record advocating screening for hypercholesterolemia and urging aggressive treatment for those at high risk. The Institute initiated a national cooperative program to that end, the National Cholesterol Education Program. For the first time, preventing coronary heart disease became a national public health goal.

In this third installment of the series, we point out that the absence of an explicit, detailed and plausible hypothesis linking hypercholesterolemia to the events in the artery wall was probably an important reason for continuing skepticism and for failure to treat elevated blood cholesterol levels. The rapid advances in understanding of lipoprotein metabolism in the 1950s and 1960s and the application of modern cellular biology in the 1970s provided the context for a modern consensus on pathogenetic mechanisms of atherogenesis.

We have previously shown that CD36 recognizes oxidation products of phospholipids on oxidized LDL (OxLDL) such as 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC). The current study was designed to examine whether the phosphocholine (PC) headgroup in POVPC constitutes an obligatory binding target for CD36. To examine the contribution of PC in the binding of POVPC to CD36, we used well-defined synthetic oxidized phospholipids (OxPLs) cross-linked to BSA or to a hexapeptide. The OxPL adducts were then tested for their ability to bind to CD36-transfected cells and for their ability to inhibit OxLDL binding to CD36. Both POVPC-BSA and POVPC-peptide adducts were high-affinity ligands for CD36 and potent inhibitors of OxLDL binding. Enzymatic removal of the entire PC moiety of the POVPC-peptide, or of the choline headgroup alone, as well as substitution of the choline headgroup by ethanolamine abrogated the inhibitory activity of POVPC. Interestingly, PC by itself or cross-linked to BSA did not show any intrinsic competition activity. In conclusion, our data demonstrate that the PC headgroup of OxPL alone is sufficient for binding to CD36, but only if presented in the correct conformation as in OxPL of OxLDL or as in POVPC-peptide adducts.

The first in this series of historical reviews dealt with the pioneering animal model work of Anitschkow, implicating blood cholesterol in the pathogenesis of atherosclerosis, and the pivotally important work of Gofman, providing evidence that lipoprotein-bound cholesterol was a major factor in the human disease. This second installment reviews the early lines of evidence linking hypercholesterolemia in humans to the progression of atherosclerosis and the risk of coronary heart disease. The argument is made that by 1970, the evidence was already strong enough to justify intervention to lower blood cholesterol levels if all the available lines of evidence had been taken into account. Yet, it would be almost two decades before lowering blood cholesterol levels became a national public health goal. Some of the reasons the "cholesterol controversy" continued in the face of powerful evidence supporting intervention are discussed.

This is the first of a series of reviews of the controversy that swirled around the "lipid hypothesis" of atherosclerosis for so many years. Today, in the era of the statins, there is no longer any doubt about the value of decreasing blood cholesterol levels. In fact, "the lower the better" is the position of many clinicians. However, getting to this point has been a long uphill battle marked by heated debate and sometimes violent disagreement. The history of this controversy is worth telling for its own sake and because remembering it may help us avoid similar mistakes in the future. The history of this controversy is worth telling for its own sake and because remembering it may help us avoid similar mistakes in the future., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

Hormone-sensitive lipase partially purified from adipose tissue of laying hens was markedly activated by cyclic AMP-dependent protein kinase. Activation was approximately 4-fold (ranging up to as great as 10-fold) compared with the much lower degree of activation obtained with analogous preparations from rat and human adipose tissues (59 and 86%, respectively). The partially purified preparations contained adequate endogenous protein kinase activity to effect complete activation with addition of cyclic AMP, ATP, and Mg(2+). Activation was blocked by protein kinase inhibitor (from rabbit skeletal muscle) but could be restored fully by addition of excess exogenous protein kinase (from bovine skeletal muscle). The fully activated lipase was slowly deactivated by dialysis at 4 degrees C and then rapidly and almost fully reactivated by addition of cyclic AMP and ATP-Mg(2+). Reactivation was blocked by protein kinase inhibitor. This deactivation-reactivation cycle was rapid at 23 degrees C with dialysis against charcoal and could be demonstrated repeatedly using a single preparation. The reversible deactivation of protein kinase-activated enzyme is presumed to reflect the action of a lipase phosphatase. Lipase prepared from tissue previously exposed to glucagon yielded a much smaller degree of activation than lipase prepared from tissue not exposed to the lipolytic hormone, indicating that the physiological hormone-induced activation is probably similar to or identical with the protein kinase activation demonstrated in the cell-free preparations. Under the conditions of assay used, the partially purified lipase fraction contained diglyceride, monoglyceride, and lipoprotein lipase activities. However, treatment with cyclic AMP-dependent protein kinase had virtually no effect on these lipase activities.

Feeding of phytol in large doses (2-5% by weight in the diet) led to accumulation of phytanic acid in the mouse, rat, rabbit, and chinchilla, the degree of accumulation depending upon the level of dietary intake. The relative concentration of phytanic acid, expressed as a percentage of the total fatty acids, was as high as 20-60% in liver and 30-40% in serum. Phytenic acid, which may be an intermediate in the conversion of phytol to phytanic acid, also accumulated. When phytol was withdrawn from the diet, tissue and serum concentrations of phytanic acid fell rapidly, which indicates the ability of the normal animal to metabolize phytanic acid readily. At high dosages in the diet, phytol inhibited growth and caused death within 1-4 weeks. In the mouse, dietary phytanic acid and dietary phytol fed in equivalent amounts were of comparable toxicity. Accumulation of tissue phytanic acid occurred more rapidly when phytanic acid was fed than when phytol was fed in equal amounts. In none of the animals fed either phytol or phytanic acid were there any signs of neurological defects. Histologic examination of rats fed phytol showed some fat accumulation, glycogen depletion, and karyokinesis in the liver. There were no pathologic changes in the retina or in the peripheral and central nervous system such as those described in Refsum's disease.

Glucose greatly increased total free fatty acid (FFA) esterification by Ehrlich ascites tumor cells. However, the FFA concentration of the cells was not altered. Less exogenous FFA was oxidized to CO(2) at any given extracellular FFA:albumin molar ratio when glucose was available, but increasing amounts of radioactive CO(2) were produced as the FFA:albumin molar ratio was raised, even in the presence of glucose. It is suggested that glucose, by providing either energy or an excess of triose acceptor for fatty acid esterification, stimulated FFA uptake only indirectly, by increasing the utilization of FFA subsequent to initial uptake from the medium, i.e., by increasing the turnover rate of the cellular FFA pool. Availability of glucose decreased the oxidation of endogenous lipid radioactivity and the depletion of endogenous lipid ester radioactivity. Most of the radioactivity utilized was derived from phospholipids, and depletion of phospholipid radio-activity was spared when glucose was available. Depletion of cellular total lipid ester also was spared in the presence of glucose. Availability of FFA did not decrease total glucose uptake or its oxidation to CO(2). Glucose utilization by these cells appears not to be regulated by FFA availability in the manner that Randle and coworkers described for muscle.

The fate of ingested chlorophyll-particularly of the phytol portion of the molecule-was studied. Uniformly (14)C-labeled pheophytin a (the Mg-free derivative of chlorophyll a) was prepared from an extract of tobacco leaves grown in (14)CO(2), and was administered by stomach tube to rats in which the thoracic duct had been cannulated. Only about 2% of the administered radioactivity was absorbed in 24 hr, largely into the thoracic duct lymph. Moreover, only a fraction of this lymph radioactivity was derived from phytol (i.e., was found in phytol, phytenic acid, or phytanic acid). The results indicated that not more than 1-2% of chlorophyll phytol is available for absorption by the rat. Similarly, after the administration of whole spinach or spinach extract (not labeled) to rats, only about 1% of the total phytol content was absorbed into the intestinal lymph. Nearly all of the administered phytol was found in the feces and the contents of the colon, and was still largely in the form of pheophytin. The study also indicated that little of the nonphytol portion of the chlorophyll molecule is absorbed.

Ehrlich ascites tumor cells release free fatty acids (FFA) during in vitro incubation in media that contain albumin. The released FFA are derived by lipolysis from endogenous lipid esters. Addition of glucose to the incubation medium greatly decreases the quantity of fatty acid released by the cells. Cyanide, which inhibits endogenous lipid oxidation but not lipolysis, increases the quantity of fatty acid released to media containing albumin and causes free fatty acid to accumulate in the cells in the absence of exogenous albumin. The release of fatty acid, either preformed or derived by lipolysis during prolonged incubations, occurs under conditions of net fatty acid uptake from the incubation medium. Net release of fatty acid from the cell occurs only when fatty acid-extracted albumin is present in the extracellular medium; extrapolation of the data suggests that net release will not occur under physiological conditions. It is postulated that free fatty acid uptake and release are independent processes, the direction of net fatty acid movement being determined by the relationship between cellular free fatty acid concentration (regulating efflux) and the molar ratio of free fatty acid to albumin in the extracellular medium (regulating uptake).

The metabolism of uniformly-labeled (14)C-phytol, (14)C-phytenic acid, and (14)C-phytanic acid was studied in the rat. Conversion of both phytol and phytenic acid to phytanic acid was demonstrated. Tracer doses of phytol-U-(14)C given orally were well absorbed (30-66%), and approximately 30% of the absorbed dose was converted to (14)CO(2) in 18 hr. After intravenous injection, 20% appeared in (14)CO(2) in 4 hr. Phytanic acid-U-(14)C given intravenously was oxidized at a comparable rate (22-37% in 4 hr) and was as rapidly oxidized as palmitic acid-1-(14)C (21% in 4 hr). Metabolism of these substrates was also studied in rats previously maintained on a diet containing 5% phytol by weight, which causes accumulation of phytanic acid, phytenic acid, and, to a lesser extent, phytol in blood and tissues. Despite the large body pools of preformed, unlabeled substrate in these animals, the fraction of an administered dose of phytol-U-(14)C or phytanic acid-U-(14)C converted to (14)CO(2) was not significantly diminished. These studies indicate that the rat has an appreciable capacity to degrade the highly branched carbon skeleton of phytol and its derivatives. Twenty-four hours after administration of phytol-U-(14)C, the lipid radioactivity remaining in the body was widely distributed among the tissues, highest concentrations being found in liver and adipose tissue. Four hours after intravenous administration of phytanic acid-U-(14)C, all of the major lipid classes in the liver contained radioactivity, most in triglycerides and phospholipids and least in cholesterol esters and lower glycerides. There was no demonstrable incorporation of mevalonate-2-(14)C or acetate-1-(14)C into liver phytanic acid when they were given intravenously to a rat previously fed phytol. Endogenous biosynthesis, if it occurs at all, must be extremely limited.