Postprandial cholesteryl ester transfer and high density lipoprotein composition in normotriglyceridemic non-insulin-dependent diabetic patients

Altered postprandial HDL metabolism is a possible cause of defective reverse cholesterol transport and increased cardiovascular risk in diabetic patients with a normal fasting lipoprotein profile. Ten normolipidemic, normoponderal non-insulin dependent diabetes mellitus (NIDDM) patients and seven controls received a 980 kcal meal containing 78 g lipids with 100 000 IU vitamin A. Chylomicron clearance was not different, but area under the curve (AUC) for retinyl palmitate in chylimicron-free serum (remnant clearance) was greater in patients (P < 0.02). LCAT activity increased postprandially to the same extent in both groups. In control subjects, cholesteryl ester transfer protein (CETP) activity (CETA) also increased by 20% (P < 0.01 at 6 h) in parallel with a 20% decrease in HDL2-CE (r = -0.55, P = 0.009). In NIDDM patients, on the contrary, CETA which was 35% higher in the fasting state (P < 0.005), decreased postprandially yet HDL2-CE remained unchanged. Postprandial HDL3 of controls were enriched with phospholipid (PL) (30.3 +/- 2.6% at 6 h) with respect to fasting (25.6 +/- 2.5%, P < 0.01) and to NIDDM-HDL3 (25.8 +/- 1.7% at 6 h, P < 0.01). These results show that variation in plasma CETA has little impact on HDL2-CE in NIDDH subjects. They support the concept that, in controls, the combined enrichment of HDL3 with PL, increased LCAT and CETA create the conditions for stimulation of cell cholesterol efflux and CE transfer to apo B lipoproteins. In NIDDM, because of the lesser HDL3 enrichment with PL and of the inverse trend of CETA, these conditions fail to occur, depriving the patients of a potentially efficient mechanism of unesterified cholesterol (UC) clearance, despite their strictly normal preprandial profile.     

Elevated high density lipoprotein concentrations in heart transplant recipients are related to impaired plasma cholesteryl ester transfer and hepatic lipase activity

Accelerated atherosclerosis is a major complication of heart transplantation, and is frequently associated with a dyslipoproteinemia characterized by a paradoxical increase in HDL-cholesterol concentration. To define this abnormality, the lipoprotein profiles of 25 heart transplant recipients (HTR) were analyzed and compared with those of 26 control subjects. HDL, as separated on the basis of density in 3 subfractions, were increased in concentration: HDL2: +51%, HDL3a: +29%, HDL3b: +32%. HDL2 and HDL3a displayed an enrichment in surface components, phospholipids, unesterified cholesterol and apo E, leading to an increased size compared with subfractions of similar density in the controls. The major steps of plasma HDL metabolism were investigated: cholesterol esterification (LCAT activity), cholesteryl ester transfer to apo B-containing lipoproteins (CETP) and the hepatic hydrolysis of HDL components (HL activity). We demonstrated a partial deficiency in CETP (-28%) and hepatic lipase (-36%) activities with normal LCAT activity. Correlations in total study population (HTR plus controls) evidenced negative associations between CETP activity and HDL3a concentrations and between HL activity and HDL2-cholesterol as a percent of total HDL-cholesterol. Therapeutic agents used in post transplantation treatment such as glucocorticoids and/or cyclosporine may be speculated thus to affect both CETP and HL activities and, by arresting the HDL cycle in a CE-saturated state, do decrease the efficiency of reverse cholesterol extraction at the site of the graft.     

Reduced cholesteryl ester transfer in plasma of patients with lipoprotein lipase deficiency

The net mass transfer of cholesteryl ester (CE) from high density lipoprotein (HDL) to the apolipoprotein (apo) B-containing lipoproteins, very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in plasma (cholesteryl ester transfer (CET)) from three patients lacking lipoprotein lipase (LpL) activity was significantly lower (P < 0.001) than in plasma from fasting control subjects with comparable triglyceride levels. Chylomicrons isolated from LpL-deficient fasting plasma showed the same low level of CET activity as observed in the intact plasma when combined with HDL and cholesteryl ester transfer protein (CETP)-containing d 1.063 g/ml bottom fractions from control subjects. Preincubation of chylomicrons and large triglyceride-rich lipoproteins (Sf > 400) from LpL-deficient plasma with milk LpL, however, stimulated the capacity to engage in CET 4- to 5-fold to the same level as chylomicrons and VLDL from control subjects after a fat load. Consistent with these measurements of CET activity in plasma, chylomicrons obtained from the LpL-deficient subjects after a 14-h fast had higher TG/CE ratios than chylomicrons from controls 3 h after ingesting a fat load (LpL-deficient 26.3 +/- 9.0 vs. controls 6.9 +/- 2.1; mean +/- SD). The mass of CETP did not differ in LpL-deficient and control subjects (LpL-deficient 1.03 +/- 0.22 micrograms/ml vs. controls 1.58 +/- 0.58 micrograms/ml). These studies are consistent with earlier in vitro studies showing that the actions of lipoprotein lipase and its lipolytic products are essential, for maximal cholesteryl ester transfer protein activity.     

Apolipoprotein A-I metabolism in cholesteryl ester transfer protein transgenic mice. Insights into the mechanisms responsible for low plasma high density lipoprotein levels

Expression of simian cholesteryl ester transfer protein (CETP) in C57BL/6 mice causes the animals' high density lipoprotein (HDL) levels to decrease. The purpose of these studies was to determine how CETP expression caused that reduction. Chemical analysis showed that the HDL of the CETP transgenic mice had about twice as much triglyceride and only about 60% as much cholesteryl ester as the HDL from the C57BL/6 mice. Both strains of mouse had high levels of a circulating lipase. When plasma from the mice was incubated at 37 degrees C for 5 h, the triglycerides in the HDL were hydrolyzed, and apoA-I was shed from the particle. However, apoA-I was shed from the CETP HDL more rapidly than it was shed from the C57BL/6 HDL. Because "free" apoA-I is rapidly cleared by the kidney, increased production of free apoA-I would be expected to shorten the average life span of apoA-I in the mouse. Kinetic analyses indicated that the life span of apoA-I was significantly reduced in the CETP transgenic mice. It was concluded that CETP expression enriched the core of the HDL with triglyceride, which rendered it vulnerable to lipolysis, causing apoA-I to be shed from the particle. That shortened the life span of apoA-I in the CETP mice, which led to lower plasma levels of the protein.     

Evidence that cholesteryl ester transfer protein-mediated reductions in reconstituted high density lipoprotein size involve particle fusion

It is well established that cholesteryl ester transfer protein (CETP) changes the size of high density lipoproteins (HDL) during incubation in vitro. It has been suggested that HDL.CETP.HDL ternary complex formation is involved in these changes. The present results, which are consistent with CETP changing the size of spherical reconstituted HDL (rHDL) by a mechanism involving fusion, support the ternary complex hypothesis. When rHDL containing a core of cholesteryl esters and either three molecules of apolipoprotein (apo) A-I/particle, (A-I)rHDL, or six molecules of apoA-II/particle, (A-II)rHDL, were incubated individually with CETP, their respective diameters decreased from 9.4 to 7.8 nm and from 9.8 to 8.8 nm. The small (A-I)rHDL and (A-II)rHDL contained, respectively, two molecules of apoA-I/particle and four molecules of apoA-II/particle. As all of the rHDL lipids and apolipoproteins were quantitatively recovered at the end of the incubations, it was apparent that there was a 50% increase in the number of particles. This increase in the number of particles can be explained as follows: (i) sequential binding of two rHDL to CETP to generate a ternary complex, (ii) fusion of the rHDL in the ternary complex, and (iii) rearrangement of the fusion product into three small particles. Various spectroscopic techniques were used to show that the small rHDL were structurally distinct from the original rHDL. These results provide the first evidence that CETP mediates the fusion of spherical rHDL.     

Cholesteryl ester transfer protein and high density lipoprotein responses to cholesterol feeding in men: relationship to apolipoprotein E genotype

Pulsed-field gel electrophoresis (PFGE) was used to study a cluster of molecular markers in the soybean genome. There were 550 kb per centimorgan (cM) in the cluster, which is close to the calculated average for the whole genome. The analysis was complicated by the presence of duplicated sequences, and some ambiguities arising from this were resolved by using second-dimension conventional electrophoresis to relate physical maps to the RFLP map of soybean. The results show that there is a high degree of conservation of 'rare cutter' sites between homoeologous regions. Finally, PFGE can confirm physical linkage of monomorphic copies of markers, which can aid in the study and comparison of homoeologous regions that are invisible to RFLP analysis.     

Human cholesteryl ester transfer protein measured by enzyme-linked immunosorbent assay with two monoclonal antibodies against rabbit cholesteryl ester transfer protein: plasma cholesteryl ester transfer protein and lipoproteins among Japanese hypercholesterolemic patients

Plasma cholesteryl ester transfer protein (CETP) concentrations were measured in Japanese subjects by an ELISA with two different monoclonal antibodies that were raised against rabbit CETP and cross-reacted against human CETP. Among 63 patients who consecutively underwent coronary angiography, the plasma CETP of 37 patients with luminal stenosis > or = 50% in their coronary arteries was not significantly different from that of the 26 patients with luminal stenosis < 50%. No other lipoprotein-related measurement except HDL-cholesterol differentiated the two groups. Among 40 hypercholesterolemic patients, no lipoprotein-related measurement other than LDL-cholesterol was found to positive correlate with the CETP. Before and after the treatment of 23 patients with simvastatin 5 mg a day for 4 weeks, plasma CETP markedly decreased in those whose pretreatment CETP was > or = 3 mg/L; no change was observed for those with lower pretreatment CETP. In the former group, negative correlation between CETP and HDL-cholesterol was demonstrated only in the posttreatment plasma.     

Dietary pectin with high viscosity lowers plasma and liver cholesterol concentration and plasma cholesteryl ester transfer protein activity in hamsters

The aim of this retrospective cohort study was to investigate whether survival of patients with breast cancer has changed over the period 1975-89. A total of 2604 women diagnosed as having invasive breast cancer at a clinical oncology unit in London were followed up for between 5 and 20 years. Patients were divided into four groups according to menstrual status (pre or post) and the staging of cancer (operable or inoperable). For each group, survival from diagnosis was compared between three consecutive 5-year cohorts, both with and without adjustments made for relevant prognostic factors. No temporal patterns were found in patients with inoperable cancer, in whom the survival rate was consistently low. Of women with operable cancers, differences were seen only among post-menopausal women, for whom the best survival patterns were seen in patients diagnosed between 1985-89. This is probably due to tamoxifen being commonly prescribed as adjuvant treatment for this cohort of patients. We cannot explain an apparently worse survival in the group of patients presenting in the early 1980s compared with that observed in the late 1970s.     

Influence of apolipoprotein composition of high density lipoprotein particles on cholesteryl ester transfer protein activity. Particles containing various proportions of apolipoproteins AI and AII

The effect of apolipoprotein (apo) composition of high density lipoproteins (HDL) on cholesteryl ester transfer protein (CETP) activity was studied by measuring the rate of radiolabeled cholesteryl esters transferred between low density lipoproteins (LDL) and HDL3 which contained various proportions of apoAI and apoAII. Ultracentrifugally isolated HDL3, which contained virtually only apoAI and apoAII in their protein moiety, were progressively enriched with apoAII upon the incubation with increasing amounts of delipidated HDL apolipoproteins. The substitution of apoAII for apoAI in HDL3 did not induce marked alteration of the lipid composition of the lipoprotein particles. The rates of cholesteryl ester exchanges with LDL in the presence of purified human CETP were significantly reduced with apoAII-enriched HDL3 as compared with non-enriched homologous particles. Consistent results were obtained by determining the rate of cholesteryl esters transferred either from LDL toward HDL3, or in the opposite direction, from HDL3 to LDL. The effect of the apoAI and apoAII content of HDL particles on CETP activity was also investigated by measuring the rate of cholesteryl esters transferred from LDL to plasma HDL3 particles which contained either only apoAI, HDL3-AI, or both apoAI and apoAII, HDL3-AIAII. HDL3-AI and HDL3-AIAII particles were isolated from human plasma by a sequential procedure which combined ultracentrifugation and anti-apoAII immunoaffinity chromatography. As observed with HDL3 artificially enriched with apoAII, cholesteryl ester transfer rates were significantly lower with plasma HDL3-AIAII than with plasma HDL3-AI particles. Kinetic analysis of the interaction of CETP with apoAII-enriched HDL3 revealed that apoAII could act as an uncompetitive inhibitor of the cholesteryl ester transfer reaction. Since the plasma levels of HDL-AI, HDL-AIAII, and HDL-AII may undergo significant physiological fluctuation, the present study suggests that HDL apoproteins may be important factors in modulating cholesteryl ester transfer rates in vivo.     

Decreased early atherosclerotic lesions in hypertriglyceridemic mice expressing cholesteryl ester transfer protein transgene

The human cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl ester from HDL to triglyceride-rich lipoproteins. The activity of CETP results in a reduction in HDL cholesterol levels, but CETP may also promote reverse cholesterol transport. Thus, the net impact of CETP expression on atherogenesis is uncertain. The influence of hypertriglyceridemia and CETP on the development of atherosclerotic lesions in the proximal aorta was assessed by feeding transgenic mice a high cholesterol diet for 16 wk. 13 out of 14 (93%) hypertriglyceridemic human apo CIII (HuCIII) transgenic (Tg) mice developed atherosclerotic lesions, compared to 18 out of 29 (62%) controls. In HuCIII/CETPTg, human apo AI/CIIITg and HuAI/CIII/CETPTg mice, 7 of 13 (54%), 5 of 10 (50%), and 5 of 13 (38%), respectively, developed lesions in the proximal aorta (P < .05 compared to HuCIIITg). The average number of aortic lesions per mouse in HuCIIITg and controls was 3.4 +/- 0.8 and 2.7 +/- 0.6, respectively in HuCIII/CETPTg, HuAI/CIIIg, and HuAI/CIII/CETPTg mice the number of lesions was significantly lower than in HuCIIITg and control mice: 0.9 +/- 0.4, 1.5 +/- 0.5, and 0.9 +/- 0.4, respectively. There were parallel reductions in mean lesion area. In a separate study, we found an increased susceptibility to dietary atherosclerosis in nonhypertriglyceridemic CETP transgenic mice compared to controls. We conclude that CETP expression inhibits the development of early atherosclerotic lesions but only in hypertriglyceridemic mice.     

Role of female sex steroids in regulating cholesteryl ester transfer protein in transgenic mice

The role of sex steroids in the regulation of cholesteryl ester transfer protein (CETP) was examined in the following groups of female transgenic mice carrying the human CETP gene: (1) normal, (2) ovariectomized, (3) ovariectomized and treated with estrogen; (4) ovariectomized and treated with progesterone; (5) ovariectomized and treated with both hormones, and (6) ovariectomized and treated with tamoxifen. CETP activity was measured in the plasma, and in the particulate and the soluble fractions of liver, muscle, and adipose tissue. Human CETP specific activity was determined by taking the difference of cholesterol ester transfer in the presence and absence of an antibody (TP2) against human CETP Ovariectomy reduced hormone levels, but did not completely abolish them from the circulation. Plasma CETP activity was significantly reduced in the tamoxifen group. There were significant reductions in CETP in liver homogenate and the soluble fraction, as well as in the particulate fraction of adipose with ovariectomy. Hormone replacement did not restore CETP activity in either the plasma or the tissues. Tamoxifin treatment resulted in a decrease in CETP activity in both fractions of liver, but had no effect on adipose. In the soluble fraction of adipose tissue and both fractions of muscle, only trace CETP activity was detected. We conclude that (1) minimal amounts of sex steroid hormones may be sufficient to affect CETP expression; (2) the effects of sex steroid hormones vary among tissues; and (3) in addition to the sex steroids, factor(s) from the ovary are needed for the full expression of CETP in this animal model.     

Decrease of hepatic triglyceride lipase levels and increase of cholesteryl ester transfer protein levels in patients with primary biliary cirrhosis: relationship to abnormalities in high-density lipoprotein

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90-95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyrylcholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. The pH optimum of the peptidase was in the range of 7.5-9.5 and the divalent cations Co2+, Mn2+, and Zn2+ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co2+, Mn2+, and Zn2+ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited alpha-chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.     

Opposite effects of cholesteryl ester transfer protein and phospholipid transfer protein on the size distribution of plasma high density lipoproteins. Physiological relevance in alcoholic patients

The aim of the present study was to investigate the role of the cholesteryl ester transfer protein (CETP) and the phospholipid transfer protein (PLTP) in determining the size distribution of high density lipoproteins (HDL) in human plasma. Whereas both purified CETP and PLTP preparations were able to promote the size redistribution of isolated HDL3, CETP favored the emergence of small HDL, while PLTP induced the formation of both small and large conversion products. When the total plasma lipoprotein fractions isolated from nine distinct subjects were incubated for 24 h at 37 degrees C with either purified PLTP or purified CETP, significant alterations in the relative proportions of the five distinct plasma HDL subpopulations, i.e., HDL2b (9.71-12.90 nm), HDL2a (8.77-9.71 nm), HDL3a (8.17-8.77 nm), HDL3b (7.76-8.17 nm), and HDL3c (7.21-7. 76 nm) were also observed. PLTP induced a significant increase in the relative abundance of HDL2b (8.66 +/- 2.34% versus 7.87 +/- 1. 83% in controls; p < 0.01) and a significant decrease in the relative abundance of HDL3a (32.76 +/- 3.42% versus 37.87 +/- 2.62% in controls; p < 0.05). In contrast, CETP significantly reduced the relative proportion of HDL2a (33.03 +/- 2.53% versus 37.56 +/- 6.43% in controls; p < 0.01) but significantly increased the relative proportion of both HDL3b (21.36 +/- 6.97% versus 15.58 +/- 7.75% in controls; p < 0.01) and HDL3c (3.21 +/- 4.84% versus 1.13 +/- 0.56% in controls; p < 0.05). Finally, in order to assess further the physiological relevance of in vitro observations, CETP activity, PLTP activity, and HDL size distribution were determined in plasmas from 33 alcoholic patients entering a cessation program. Alcohol withdrawal was associated with (i) a significant increase in plasma CETP activity (173.5 +/- 70.5%/h/ml before versus 223.2 +/- 69. 3%/h/ml after alcohol withdrawal, p = 0.0007), (ii) a significant reduction in plasma PLTP activity (473.9 +/- 203.7%/h/ml before versus 312.7 +/- 148.4%/h/ml after alcohol withdrawal, p = 0.0001), and (iii) a significant shift of large HDL2b and HDL2a toward small HDL3b and HDL3c. On the one hand, changes in plasma CETP activity correlated negatively with changes in the proportion of HDL2a (r = -0.597, p = 0.0002) and positively with changes in the proportion of HDL3b (r = 0.457, p = 0.0075). On the other hand, changes in plasma PLTP activity correlated positively with changes in the proportion of HDL2b (r = 0.482, p = 0.0045) and negatively with changes in the proportion of HDL3a (r = -0.418, p = 0.0154). Taken together, data of the present study revealed that plasma PLTP and CETP can exert opposite effects on the size distribution of plasma HDL. PLTP can promote the formation of HDL2b particles at the expense of HDL3a, while CETP can promote the formation of HDL3b particles at the expense of HDL2a.     

Plasma cholesteryl ester transfer protein activity in hyper- and hypothyroidism

Thyroid dysfunction is associated with multiple changes in lipoprotein metabolism, and we have determined the effects of thyroid dysfunction on plasma cholesteryl ester transfer protein (CETP) activity. CETP is a plasma protein that mediates the exchange of cholesteryl ester and triglyceride between plasma lipoproteins and plays an important role in high-density lipoprotein metabolism and in the reverse cholesterol transport pathway. Plasma CETP activity was assayed in 18 hyperthyroid and in 17 hypothyroid patients, before and after treatment, by measuring the transfer of cholesteryl esters from exogenous radiolabeled high-density lipoprotein to apolipoprotein B-containing lipoproteins. Plasma CETP activity was increased in hyperthyroid patients, compared with their matched controls (22.11 +/- 8.92% transferred/5 microL.4 h vs. 16.75 +/- 6.48, P < 0.05), whereas in hypothyroid patients, plasma CETP activity was decreased (11.14 +/- 4.84% transferred/5 microL.4 h vs. 17.26 +/- 7.13, P < 0.01). Plasma CETP activity decreased after treatment of thyrotoxicosis, although a significant change was observed, mainly in the severely thyrotoxic patients with free T4 > 100 pmol/L (n = 11, 25.61 +/- 8.12% transferred/5 microL.4 h vs. 21.71 +/- 7.84, P < 0.05). In the hypothyroid patients, there was a significant increase in plasma CETP activity after thyroxine replacement (11.14 +/- 4.84% transferred/5 microL.4 h vs. 15.46 +/- 6.71, P < 0.01). There was a strong positive correlation between log(free T4) and plasma CETP activity (r = 0.51, P < 0.001). In summary, both hyper- and hypothyroidism are associated with significant changes in plasma CETP activity, and these changes are corrected when the patients have been rendered euthyroid.     

Plasma cholesteryl ester transfer activity is modulated by the phase transition of the lipoprotein core

Previous studies have shown that lipid transfer protein (LTP) activity is strongly temperature dependent, demonstrating a dramatic rise in activity near 37 degrees C. We have investigated the origin of this rapid rise in LTP activity. LTP-mediated transfers of radiolabeled cholesteryl ester (CE) from LDL to HDL, HDL to LDL, LDL to biotin-LDL, HDL to biotin-HDL, and between liposomes were determined as a function of assay temperature. Only assays containing LDL demonstrated this rapid rise in CE transfer activity. In contrast, TG transfer was almost linear with assay temperature. As human LDL CE undergoes a thermal phase transition near 37 degrees C, we investigated whether the rapid rise in CE transfer was dependent on this transition. Monkey LDL were isolated from animals consuming diets containing cholesterol and enriched in saturated, monounsaturated, or polyunsaturated fatty acids. With these LDL as substrate, the CE transfer between 21 degrees and 49 degrees C could be described by two straight lines, the intersection of which defined the inflection temperature. Among eight LDL samples, the inflection temperature was highly correlated with the CE phase transition determined by differential scanning calorimetry (r2 = 0.86). Both calorimetry and CE transfer activity inflection values were correlated with the saturated + monoene/polyene ratio of the LDL cholesteryl esters (r2 = 0.733 and 0.612, respectively). For LDL with inflection temperatures below 37 degrees C, CE transfer activity at 37 degrees C increased 10-14% for each 1 degree C decrease in the inflection temperature. We conclude that LTP activity is markedly affected by the physical state of the core CE. Diets rich in saturated fatty acids may result in LDL that are poor LTP substrates, which may hinder LTP's ability to promote normal lipoprotein remodeling.     

Cholesteryl ester transfer protein activity enhances plasma cholesteryl ester formation. Studies in CETP transgenic mice and human genetic CETP deficiency

The plasma cholesteryl ester transfer protein (CETP) promotes the removal of HDL cholesteryl esters and is thought to stimulate reverse cholesterol transport (RCT). However, mechanisms by which CETP may stimulate RCT are poorly understood. Thus, we examined the relationship between plasma CETP expression and plasma cholesteryl ester formation in CETP transgenic (Tg) mice, hamsters, and human subjects with genetic CETP deficiency. Incubation of CETP Tg mouse plasma showed a 20% to 40% increase in plasma cholesterol esterification rate (CER, P < .05) compared with control mice. Injection of a neutralizing CETP monoclonal antibody (MAb) (TP2) into natural flanking region CETP Tg mice resulted in an increase in plasma free cholesterol (FC) concentration, FC/CE ratio, FC/phosphatidylcholine ratio, and hepatic CETP mRNA. In hamsters, CETP inhibition also resulted in an increase in plasma FC/phosphatidylcholine ratio and increased CETP mRNA in adipose tissue. In humans with two common CETP gene mutations (an intron 14 splicing defect and a D442G missense mutation), mean plasma CERs were 39 and 60, respectively, compared with 89 nmol x mL-1 x h-1 in normal subjects. By contrast, lecithin:cholesterol acyltransferase (LCAT) mass was normal in CETP-deficient subjects. MAb neutralization of CETP activity in incubated human plasma did not alter the LCAT reaction, even after supplementation with discoidal HDL and VLDL. Thus, genetic alterations in CETP levels lead to secondary changes in the plasma LCAT reaction, possibly because of remodeling of HDL by CETP acting in concert with other factors in vivo. In human genetic CETP deficiency, a moderate impairment in the plasma LCAT reaction may contribute to a defect in RCT, providing a potential mechanism to explain the recently observed excess of coronary heart disease in these subjects.     

Inhibition of lipoprotein lipase induced cholesterol ester accumulation in human hepatoma HepG2 cells

WD-repeat proteins contain four to eight copies of a conserved motif that usually ends with a tryptophan-aspartate (WD) dipeptide. The Saccharomyces cerevisiae PWP2 gene, identified by sequencing of chromosome III, is predicted to contain eight so-called WD-repeats, flanked by nonhomologous extensions. This gene is expressed as a 3.2-kb mRNA in all cell types and encodes a protein of 104 kDa. The PWP2 gene is essential for growth because spores carrying the pwp2 delta 1::HIS3 disruption germinate before arresting growth with one or two large buds. The growth defect of pwp2 delta 1::HIS3 cells was rescued by expression of PWP2 or epitope-tagged HA-PWP2 using the galactose-inducible GALI promoter. In the absence of galactose, depletion of Pwp2p resulted in multibudded cells with defects in bud site selection, cytokinesis, and hydrolysis of the septal junction between mother and daughter cells. In cell fractionation studies, HA-Pwp2p was localized in the particulate component of cell lysates, from which it would be solubulized by high salt and alkaline buffer but not by nonionic detergents or urea. Indirect immunofluorescence microscopy indicated that HA-Pwp2p was clustered at multiple points in the cytoplasm. These results suggest that Pwp2p exists in a proteinaceous complex, possibly associated with the cytoskeleton, where it functions in control of cell growth and separation.