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Gram-negative sepsis is a major death cause in intensive care units. Accumulating evidence indicates the protective role of plasma lipoproteins such as high-density lipoprotein (HDL) in sepsis. It has recently been shown that septic HDL is almost depleted from apolipoprotein CI (apoCI), suggesting that apoCI may be a protective factor in sepsis. Sequence analysis revealed that apoCI possesses a highly conserved consensus KVKEKLK binding motif for lipopolysaccharide (LPS), an outer-membrane component of gram-negative bacteria. Through avid binding to LPS involving this motif, apoCI improved the presentation of LPS to macrophages in vitro and in mice, thereby stimulating the inflammatory response to LPS. Moreover, apoCI dose-dependently increased the early inflammatory response to Klebsiella pneumoniae-induced pneumonia, reduced the number of circulating bacteria, and protected mice against fatal sepsis. Our data support the hypothesis that apoCI is a physiological protector against infection by enhancing the early inflammatory response to LPS and suggest that timely increase of apoCI levels could be used to efficiently prevent and treat early sepsis.

Previous studies have shown that overexpression of human apolipoprotein C-I (apoC-I) results in moderate hypercholesterolemia and severe hypertriglyceridemia in mice in the presence and absence of apoE. We assessed whether physiological endogenous apoC-I levels are sufficient to modulate plasma lipid levels independently of effects of apoE on lipid metabolism by comparing apolipoprotein E gene-deficient/apolipoprotein C-I gene-deficient (apoe-/-apoc1-/-), apoe-/-apoc1+/-, and apoe-/-apoc1+/+ mice. The presence of the apoC-I gene-dose-dependently increased plasma cholesterol (+45%; P < 0.001) and triglycerides (TGs) (+137%; P < 0.001), both specific for VLDL. Whereas apoC-I did not affect intestinal [3H]TG absorption, it increased the production rate of hepatic VLDL-TG (+35%; P < 0.05) and VLDL-[35S]apoB (+39%; P < 0.01). In addition, apoC-I increased the postprandial TG response to an intragastric olive oil load (+120%; P < 0.05) and decreased the uptake of [3H]TG-derived FFAs from intravenously administered VLDL-like emulsion particles by gonadal and perirenal white adipose tissue (WAT) (-34% and -25%, respectively; P < 0.05). As LPL is the main enzyme involved in the clearance of TG-derived FFAs by WAT, and total postheparin plasma LPL levels were unaffected, these data demonstrate that endogenous apoC-I suffices to attenuate the lipolytic activity of LPL. Thus, we conclude that endogenous plasma apoC-I increases VLDL-total cholesterol and VLDL-TG dose-dependently in apoe-/- mice, resulting from increased VLDL particle production and LPL inhibition.

Objective: To investigate associations of gene polymorphisms of the apoE-CI-CII gene cluster and the LDL-R gene on coronary artery disease (CAD) and their interactions with alcohol drinking and smoking in the Chinese Han population., Methods: A questionnaire survey of the behaviors of smoking and drinking, dietary patterns and anamnesis was conducted among 203 patients of CAD, aged 65.0 +/- 11.1 years, and 365 controls, aged 63.6 +/- 12.0 years. Peripheral blood samples were colleted and the total DNA was extracted. The apoE genotypes were identified by multiplex amplification refractory mutation system (multi-AMRS), the apoCI promoter polymorphisms and AvaII polymorphisms of the apoCII and LDL-R gene were detected by using PCR-RFLP. Pairwise linkage disequilibrium coefficients (D, D') were estimated by the LINKAGE program. The interactions between genes with alcohol drinking and smoking were analyzed by using multivariate logistic regression models., Results: The differences of systolic/diastolic blood pressure, total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) concentrations, smoking and drinking were significant between subjects with CAD and controls. The frequencies of apoE gene epsilon 3/4 genotype (25.9%) and epsilon 4 (13.9%) in CAD were significantly higher than those in controls (12.5% and 6.9%, respectively, p < 0.05). A significant difference was also found for the apoCI locus, the frequencies of H2 allele were 20.5% in the CAD and 11.3% in the control. Linkage disequilibrium coefficient D' was 0.672 (p < 0.01) between apoE and apoCI genes. Significant differences for a deficit of epsilon 3-H1-T1 and excess of epsilon 4-H2-T1 was found in CAD by estimation of the haplotype frequencies. After control for possible confounding factors, the multivariate logistic analysis showed that epsilon 4, H2 allele, smoking and drinking were risk factors of CAD. A significant interaction among epsilon 4, H2 and smoking was observed (OR 18.3, 95% CI: 2.35-150.81, p < 0.05), it was a multiplicative model. An additive model was shown among epsilon 4, H2 and drinking (OR12.7, 95% CI: 2.8-58.6, p < 0.05)., Conclusion: The results suggested that both apoE and apoCI on chromosome 19 were the susceptibility locus for CAD, their linkage disequilibrium should be responsible for the development of CAD. Drinking and smoking enhance the genetic predisposition to CAD.

Plasma cholesteryl ester transfer protein (CETP) has a profound effect on neutral lipid transfers between HDLs and apolipoprotein B (apoB)-containing lipoproteins when it is expressed in combination with human apoA-I in HuAI/CETP transgenic (Tg) rodents. In the present study, human apoA-I-mediated lipoprotein changes in HuAI/CETPTg rats are characterized by 3- to 5-fold increments in the apoB-containing lipoprotein-to-HDL cholesterol ratio, and in the cholesteryl ester-to-triglyceride ratio in apoB-containing lipoproteins. These changes occur despite no change in plasma CETP concentration in HuAI/CETPTg rats, as compared with CETPTg rats. A number of HDL apolipoproteins, including rat apoA-I and rat apoC-I are removed from the HDL surface as a result of human apoA-I overexpression. Rat apoC-I, which is known to constitute a potent inhibitor of CETP, accounts for approximately two-thirds of CETP inhibitory activity in HDL from wild-type rats, and the remainder is carried by other HDL-bound apolipoprotein inhibitors. It is concluded that human apoA-I overexpression modifies HDL particles in a way that suppresses their ability to inhibit CETP. An apoC-I decrease in HDL of HuAI/CETPTg rats contributes chiefly to the loss of the CETP-inhibitory potential that is normally associated with wild-type HDL.

We have recently shown that the predominant hypertriglyceridemia in human apolipoprotein C1 (APOC1) transgenic mice is mainly explained by apoCI-mediated inhibition of the lipoprotein lipase (LPL)-dependent triglyceride (TG)-hydrolysis pathway. Since the very-low-density lipoprotein receptor (VLDLr) and apoCIII are potent modifiers of LPL activity, our current aim was to study whether the lipolysis-inhibiting action of apoCI would be dependent on the presence of the VLDLr and apoCIII in vivo. Hereto, we employed liver-specific expression of human apoCI by using a novel recombinant adenovirus (AdAPOC1). In wild-type mice, moderate apoCI expression leading to plasma human apoCI levels of 12-33 mg/dl dose-dependently and specifically increased plasma TG (up to 6.6-fold, P < 0.001), yielding the same hypertriglyceridemic phenotype as observed in human APOC1 transgenic mice. AdAPOC1 still increased plasma TG in vldlr(-/-) mice (4.1-fold, P < 0.001) and in apoc3(-/-) mice (6.8-fold, P < 0.001) that were also deficient for the low-density lipoprotein receptor (LDLr) and LDLr-related protein (LRP) or apoE, respectively. Thus, irrespective of receptor-mediated remnant clearance by the liver, liver-specific expression of human apoCI causes hypertriglyceridemia in the absence of the VLDLr and apoCIII. We conclude that apoCI is a powerful and direct inhibitor of LPL activity independent of the VLDLr and apoCIII.

Genetically engineered mice demonstrated that apolipoprotein (apo) CI is a potent, physiological inhibitor of plasma cholesteryl ester transfer protein (CETP) activity. The goal of this study was to determine the molecular mechanism of the apoCI-mediated blockade of CETP activity. Kinetic analyses revealed that the inhibitory property of apoCI is independent of the amount of active CETP, but it is tightly dependent on the amount of high density lipoproteins (HDL) in the incubation mixtures. The electrostatic charge of HDL, i.e. the main carrier of apoCI in human plasma, is gradually modified with increasing amounts of apoCI, and the neutralization of apoCI lysine residues by acetylation produces a marked reduction in its inhibitory potential. The inhibitory property of full-length apoCI is shared by its C-terminal alpha-helix with significant electrostratic properties, whereas its N-terminal alpha-helix with no CETP inhibitory property has no effect on HDL electronegativity. Finally, binding experiments demonstrated that apoCI and to a lower extent its C-terminal alpha-helix are able to disrupt CETP-lipoprotein complexes in a concentration-dependent manner. It was concluded that the inhibition of CETP activity by apoCI is in direct link with its specific electrostatic properties, and the apoCI-mediated reduction in the binding properties of lipoproteins results in weaker CETP-HDL interactions and fewer cholesteryl ester transfers.

Context: Low birth weight is associated with increased cardiovascular disease in adulthood, and differences in the molecular weight, composition, and quantity of lipoprotein subclasses are associated with coronary artery disease., Objective: To determine if there are novel patterns of lipoprotein heterogeneity in low-birth-weight infants., Design, Setting, and Participants: Prospective study at a US medical center of a representative sample of infants (n = 163; 70 white and 93 black) born at 28 or more weeks of gestational age between January 3, 2000, and September 27, 2000. This sample constituted 20% of all infants born during the study period at this site., Main Outcome Measures: Plasma levels and particle sizes of lipoprotein subclasses and plasma concentrations of lipids, lipoproteins (high-density lipoprotein [HDL] and low-density lipoprotein [LDL]), and apolipoproteins., Results: An elevated lipoprotein peak of a particle with density between 1.062 and 1.072 g/mL was identified using physical-chemical methods. This subclass of large HDL was enriched in apolipoprotein C-I (apo C-I). Based on the amount of the apo C-I-enriched HDL peak, 156 infants were assigned to 1 of 4 groups: 0 (none detected), 17%; 1 (possibly present), 41%; 2 (probably present), 22%; 3 (elevated), 19%. Infants in group 3, compared with those in the other 3 groups, had significantly (P<.001) lower mean birth weight (2683.7 vs 3307.1 g) and younger mean gestational age (36.2 vs 39.3 wk). After correction for age, infants in group 3 had significantly higher levels of total and large HDL cholesterol and of total and large LDL cholesterol and LDL particle number. However, infants in group 3 had lower levels of small HDL, very low-density lipoproteins, and triglycerides than infants in the other 3 groups. This lipoprotein profile differed from that in infants born small for gestational age, who had significantly higher triglyceride (P<.001) and apo B (P = .04) levels, but lower levels of total and large HDL cholesterol (P<.001) and apo A-I (P<.001)., Conclusions: Because apo C-I-enriched HDL, and purified apo C-I alone, promotes apoptosis in vitro, increased amounts of this particle may have physiological significance and identify a novel group of low-birth-weight infants apparently distinct from traditionally classified small-for-gestational-age infants.

We examined the effect of APOC1-317insCGTT allele status (HpaI RFLP, deletion [H1] and insertion [H2] alleles) on serum apolipoprotein (apo) C-I level in 362 Hispanic children in the Columbia University BioMarkers Study. The H2 allele was present in 147 subjects (40.6%). Serum apoC-I was 20% lower in the presence of the H2 allele in APOE epsilon3/epsilon3 homozygotes (P=0.003) but did not differ by H2 status in epsilon4 carriers. Insufficient numbers of epsilon2 carriers (N=45) were present for analysis. In multivariate analysis in the epsilon3/epsilon3 context, after adjusting for potential covariate effects and familial aggregation, the mean effect of H2/* versus H1/H1 on apoC-I level, was estimated to be 2.15+/-0.55mg/dl (P<0.0025). Plasma triglyceride level was weakly correlated with serum apoC-I level (Pearson's r=0.17, P<0.001) but was highly correlated with serum apoC-III (Pearson's r=0.74, P<0.0001). Nevertheless, presence of the H2 allele was not significantly associated with serum apoC-III level. Thus, the effect of APOC1 genotype on serum apoC-I level was not due to apoC-I level serving as a surrogate for triglyceride level. The APOC1-317insCGTT allele is a commonly polymorphic genetic marker that is associated with serum apoC-I level in the APOE epsilon3/epsilon3 context. These findings suggest that the mechanism of the previously described association with plasma TG is, at least in part, related to the correlation of the polymorphism with the level of expression of apoC-I.

The lack of a cell culture system to support hepatitis C virus (HCV) replication has hampered studies of this frequent cause of chronic liver disease. However, pseudotyped retroviral particles (pp) bearing the HCV envelope glycoproteins have provided a different approach to HCV studies. We used genotype 1a pp to detect neutralizing antibodies (NtAb) in eight chimpanzees and four humans infected with 1a strains, and developed pp of genotypes 2a, 3a, 4a, 5a, and 6a to study crossreactivity. NtAb was detected in one of four chimpanzees and none of three humans with acute resolving infection, suggesting that NtAb is not required for HCV clearance. NtAb were detected at high titer in two of four chimpanzees and, in Patient H, all with persistent infection; responses paralleled humoral responses to envelope 1 and 2 proteins and, in some cases, correlate also with antibodies to the hypervariable region 1, previously thought to be the primary site of neutralization. NtAb raised during 1a infections could neutralize HCVpp of genotypes 4a, 5a, and 6a but had only limited reactivity against 2a and 3a. The detection of high-titer NtAb with cross-genotype reactivity has important implications for the development of active and passive immune-prophylaxis strategies against HCV. Finally, we found that HCVpp infectivity was enhanced by human or chimpanzee sera; apolipoprotein C1 alone or as a component of high-density lipoproteins caused this enhancement. Future studies of the in vivo role of apolipoprotein C1 might provide additional insights into the infection process of HCV.

ApoC-I plays an important role in controlling plasma lipid metabolism, however little is known about factors regulating the hepatic synthesis and secretion of this apolipoprotein. In the present study, we have carried out experiments with human hepatoma (HepG2) cells, in order to determine the effect of different tissue culture conditions on cellular lipid levels and on the production of apoC-I (and apoE) at the protein and mRNA level. Cells incubated for 48 h with 10% human serum had significantly higher cellular triglyceride (22%, P<0.05) and cholesterol levels (19%, P<0.01), higher medium apoC-I and apoE levels (2.6- and 2.9-fold, respectively), but similar levels of apoC-I and apoE mRNA, compared to cells incubated with 10% human lipoprotein-deficient serum (LPDS). Serum containing only HDL, or containing HDL with LDL, also increased cellular lipids and increased secreted apoC-I and apoE levels without altering apoC-I and apoE mRNA levels. Incubation of cells with Intralipid triglyceride (625 microM), increased cellular triglyceride (2.8-fold, P<0.001), decreased cellular cholesterol (32%, P<0.01), decreased cellular and medium apoC-I (24 and 26%, P<0.01) and had no effect on apoC-I mRNA levels. Additional experiments in which cells were loaded with cholesterol (incubation with 10 microg/ml cholesterol plus 1 microg/ml 25-hydroxycholesterol) or depleted of cholesterol (statin treatment) confirmed that secretion of apoC-I by HepG2 cells was dependent on cellular cholesterol levels and independent of changes in apoC-I mRNA levels. These results demonstrate that cellular cholesterol rather than triglyceride levels play a role in controlling apoC-I production by HepG2 cells and that this regulation occurs at a post-transcriptional level.

Early diagnosis and immediate therapeutic interventions are crucial factors to reduce the damage extent and the risk of death. Currently, the diagnosis of stroke relies on neurological assessment of the patient and neuro-imaging techniques including computed tomography and/or magnetic resonance imaging scan. An early diagnostic marker of stroke, ideally capable to discriminate ischemic from hemorrhagic stroke would considerably improve patient acute management. Using surface-enhanced laser desorption/ionization (SELDI) technology, we aimed at finding new early diagnostic plasmatic markers of stroke. Strong anionic exchange (SAX) SELDI profiles of plasma samples from 21 stroke patients were compared to 21 samples from healthy controls. Seven peaks appeared to be differentially expressed with significant p values (p < 0.05). Proteins were stripped from the SAX chips, separated on a one-dimensional electrophoresis (1-DE) gel and stained using mass spectrometry (MS)-compatible silver staining. Following in-gel tryptic digestion, the peptides were analyzed by MS. Four candidate proteins were identified as apolipoprotein CI (ApoC-I), apolipoprotein CIII (ApoC-III), serum amyloid A (SAA), and antithrombin-III fragment (AT-III fragment). Assessment of ApoC-I and ApoC-III levels in plasma samples using a sandwich enzyme-linked immunosorbent assay (ELISA) allowed to distinguish between hemorrhagic (n = 15) and ischemic (n = 16) stroke (p < 0.001). To the best of our knowledge, ApoC-I and ApoC-III are the first reported plasmatic biomarkers capable to accurately distinguish between ischemic and hemorrhagic stroke in a small number of patients. It requires further investigation in a large cohort of patients.

Objective: Apolipoprotein C-I (apoC-I) influences lipoprotein metabolism, but little is known about its cellular effects in aortic smooth muscle cells (ASMC)., Methods and Results: In cultured human ASMC, apoC-I and immunoaffinity purified apoC-I-enriched high-density lipoproteins (HDL) markedly induced apoptosis (5- to 25-fold), compared with control cells, apoC-I-poor HDL, and apolipoprotein C-III (apoC-III) as determined by 4', 6-diamidino-2-phenylindole dihydrochloride staining and DNA ladder assay. Preincubation of cells with GW4869, an inhibitor of neutral sphingomyelinase (N-SMase), blocked apoC-I-induced apoptosis, an effect that was bypassed by C-2 ceramide. The activity of N-SMase was increased 2- to 3-fold in ASMC by apoC-I, apoC-I-enriched HDL, and tumor necrosis factor alpha (TNF-alpha) (positive control) after 10 minutes and then decreased over 60 minutes, which is a kinetic pattern not seen with controls, apoC-III, and apoC-I-poor HDL. ApoC-I and apoC-I-enriched HDL stimulated the generation of ceramide, the release of cytochrome c from mitochondria, and activation of caspase-3 greater than that found in controls, apoC-III, and apoC-I-poor HDL. GW4869 inhibited apoC-I-induced production of ceramide and cytochrome c release., Conclusions: ApoC-I and apoC-I-enriched HDL activate the N-SMase-ceramide signaling pathway, leading to apoptosis in human ASMC, which is an effect that may promote plaque rupture in vivo.

Objective: To investigate into the relationship of apolipoprotein CI (ApoCI) polymorphism with sporadic Alzheimer's disease (AD) in Chinese., Subjects and Methods: A total of 257 AD patients and 242 age-matched elderly individuals were genotyped for the ApoCI HpaI and apolipoprotein E (ApoE) HhaI polymorphisms., Results: The ApoCI A allele was associated with AD of moderate to severe dementia when patients were divided into two subgroups according to Clinical Dementia Rating scale, and the AA genotype was strongly associated with moderate to severe AD in ApoE epsilon4 allele carriers [odds ratio (OR) = 8.19, 95% confidential interval: 1.28-52.30, after adjusting for age and gender by logistic regression analysis], although in total no significant differences of allele or genotype frequency between patients and controls were found., Conclusion: The present study partially confirmed the previous findings, suggesting that the ApoCI A allele might contribute to the susceptibility to moderate to severe sporadic AD in Chinese.

Obese obob mice with strong overexpression of the human apolipoprotein C1 (APOC1) exhibit excessive free fatty acid (FFA) and triglyceride (TG) levels and severely reduced body weight (due to the absence of subcutaneous adipose tissue) and skin abnormalities. To evaluate the effects of APOC1 overexpression on hepatic and peripheral insulin sensitivity in a less-extreme model, we generated obob mice with mild overexpression of APOC1 (obob/APOC1(+/-)) and performed hyperinsulinemic clamp analysis. Compared with obob littermates, obob/APOC1(+/-) mice showed reduced body weight (-25%) and increased plasma levels of TG (+632%), total cholesterol (+134%), FFA (+65%), glucose (+73%), and insulin (+49%). Hyperinsulinemic clamp analysis revealed severe whole-body and hepatic insulin resistance in obob/APOC1(+/-) mice and, in addition, increased hepatic uptake of FFA and hepatic TG content. Treatment of obob/APOC1(+/-) mice with rosiglitazone strongly improved whole-body insulin sensitivity as well as hepatic insulin sensitivity, despite a further increase of hepatic fatty acid (FA) uptake and a panlobular increase of hepatic TG accumulation. We conclude that overexpression of APOC1 prevents rosiglitazone-induced peripheral FA uptake leading to severe hepatic steatosis. Interestingly, despite rosiglitazone-induced hepatic steatosis, hepatic insulin sensitivity improves dramatically. We hypothesize that the different hepatic fat accumulation and/or decrease in FA intermediates has a major effect on the insulin sensitivity of the liver.

Background: High plasma lipids are one of the risk factor of atherosclerosis. Both environmental (diet, physic activity) and genetic factors have been implicated in the development of hyperlipidaemia. Apolipoprotein (apo) CI plays an important role in plasma cholesterol and triglycerides transport by VLDL particles. The aim of the study was to establish the role of the insertion/deletion polymorphism in apoCI gene in the determination of plasma lipids in children., Methods and Results: Using PCR and restriction analysis (HpaI) we have measured I/D polymorphism in APOCI gene in two groups of children selected from opposite ends of the cholesterol distribution curve of 2000 children. Eighty-two children in high-(HCG) and eighty-six children in low-(LCG) cholesterolemic groups participated on the study. No significant difference was found in the frequencies of the APOCI genotypes or alleles between HCG vs. LCG. Association between LDL cholesterol and genotypes within the LCG was found--the D/D homozygotes have higher lipid level compared to the others (p < 0.05). In LCG opposite, but insignificant (p = 0.09) trend was observed., Conclusions: The widespread I/D polymorphism in the gene for APOCI determines the plasma lipid levels in childhood and it could become another important genetic marker that plays a role in the genetic determination of cholesterolemia.

Numerous factors are known to affect the plasma metabolism of HDL, including lipoprotein receptors, lipid transfer protein, lipolytic enzymes and HDL apolipoproteins. In order to better define the role of HDL apolipoproteins in determining plasma HDL concentrations, the aims of the present study were: a) to compare the in vivo rate of plasma turnover of HDL apolipoproteins [i.e., apolipoprotein A-I (apoA-I), apoC-I, apoC-III, and apoE], and b) to investigate to what extent these metabolic parameters are related to plasma HDL levels. We thus studied 16 individuals with HDL cholesterol levels ranging from 0.56-1.66 mmol/l and HDL apoA-I levels ranging from 89-149 mg/dl. Plasma kinetics of HDL apolipoproteins were investigated using a primed constant (12 h) infusion of deuterated leucine. Plasma HDL apolipoprotein levels were 41.8 +/- 1.5, 9.7 +/- 0.5, 4.9 +/- 0.5, and 0.7 +/- 0.1 micromol/l for apoA-I, apoC-I, apoC-III and apoE. Plasma transport rates (TRs) were 388.6 +/- 24.7, 131.5 +/- 12.5, 66.5 +/- 9.1, and 31.4 +/- 3.3 nmol.kg-1.day-1; and residence times (RTs) were 5.1 +/- 0.4, 3.7 +/- 0.3, 3.6 +/- 0.3, and 1.1 +/- 0.1 days, respectively. HDL cholesterol and apoA-I levels were significantly correlated with HDL apoA-I RT (r = 0.69 and r = 0.56), and were not significantly correlated with HDL apoA-I TR. In contrast, HDL apoC-I, apoC-III, and apoB levels were all positively related to their TRs and not their RTs. HDL apoC-III TR was positively correlated with levels of HDL apoC-III (r = 0.73, P < 0.01), and with those of HDL cholesterol and apoA-I (r = 0.54 and r = 0.53, P < 0.05, respectively). HDL apoC-III TR was in turn related to HDL apoA-I RT (r = 0.51, P < 0.05). Together, these results provide in vivo evidence for a link between the metabolism of HDL apoC-III and apoA-I, and suggest a role for apoC-III in the regulation of plasma HDL levels.

Apolipoprotein (apo) E and C-I are plasma apolipoproteins that have been implicated in the etiology of atherosclerosis and obesity, respectively. Both proteins are synthesized and secreted by macrophages, though pharmacological regulation of their production is poorly understood. The authors compared the effect of 2 HMG-CoA reductase inhibitors, atorvastatin and cerivastatin, on the synthesis and secretion of apoE and apoC-I by THP-1 macrophages. Atorvastatin reduced medium apoE and cellular apoE mRNA of PMA-activated THP-1 cells in a dose-dependent manner (-24% and -22%, respectively, at 1-micromol/L, P < 0.01). ApoC-I in the medium was also reduced by atorvastatin in a dose-dependent manner, though to a lesser extent (-15% at 1-micromol/L, P < 0.05). Cerivastatin similarly reduced medium apoE (-20% at 1-micromol/L, P < 0.05) and cellular apoE mRNA (-31% at 1-micromol/L, P < 0.05), and significantly lowered cellular apoC-I mRNA (-15%, P < 0.05), but not apoC-I in the medium. In experiments with THP-1 macrophages loaded with cholesterol (ie, 24-hour incubation with acetyl-LDL), atorvastatin and cerivastatin (1-micromol/L) significantly (P < 0.05) reduced both medium apoE (-30% and -25%, respectively) and cellular apoE mRNA (-25% and -17%, respectively). A lower and less consistent effect was observed on medium apoC-I (-6% and -18%, respectively) and cellular apoC-I mRNA (-13% and -19%, respectively). These data demonstrate that statins have the capacity to reduce the synthesis and secretion of both apoE and apoC-I in THP-1 macrophages loaded or unloaded with cholesterol.

An Hpa I restriction site located 317 bp upstream of the transcription initiation site of the apoC-I gene has been shown to increase apoC-I gene transcription in vitro. The aim of the present study was to determine whether this genetic polymorphism was associated in vivo with increased plasma levels of apoC-I. In a cohort of French-Canadians (n=391) recruited for a family study, we found strong linkage disequilibrium between the genes for apoC-I and apoE (as reported before for European-Americans), such that the apoC-I Hpa I-negative (H1) allele was strongly associated with apoE epsilon 3, whereas the apoC-I Hpa I-positive (H2) allele was strongly associated with apoE epsilon 2 and epsilon 4. ApoC-I and apoE were measured by ELISA in total plasma and in very low-density lipoproteins (VLDL) separated by ultracentrifugation (d<1.006 g/ml), and then by difference for the non-VLDL fraction (d>1.006 g/ml), in a subset of families selected for their diverse apoE genotypes. Subjects were divided into normolipidemic (NL, n=89, TG<2.3 mmol/l, LDL-C<3.8 mmol/l) and hyperlipidemic groups (HL, n=88, TG>2.3 mmol/l and/or LDL-C>3.8 mmol/l). In NL subjects, apoC-I levels were not significantly associated with apoC-I genotype (H1/H1, H1/H2 or H2/H2). They were, however, related to apoE genotype, such that apoE3/2 subjects tended to have higher and apoE4/3 subjects tended to have lower concentrations of total plasma and non-VLDL apoC-I and apoE. Total plasma, VLDL and non-VLDL apoC-I and E levels were also higher in HL subjects with an apoE2/2 or apoE3/2 genotype. These results suggest that plasma levels of apoC-I are more strongly influenced by apoE genotype than by the Hpa I apoC-I promoter polymorphism, which probably reflects an effect of different apoE isoforms on plasma lipoprotein and plasma apoC-I metabolism, rather than a direct effect of apoE alleles on apoC-I transcription.

Background: Recent studies have suggested that apoprotein (apo) CI in very low-density lipoprotein (VLDL) plays an important role in causing hypertriglyceridemia independent of apo CIII, which is associated with coronary heart disease (CHD). Because the incidence of CHD is increased in diabetic patients and is even higher when diabetic nephropathy is developed, we measured apo CI levels in VLDL from type 2 diabetic patients, with various degree of nephropathy, and compared the results with those for healthy controls or nondiabetic patients with chronic renal failure (CRF)., Methods: This study enrolled healthy control subjects, type 2 diabetic patients with normoalbuminuria, microalbuminuria, overt proteinuria, and CRF on hemodialysis and nondiabetic hemodialysis patients. VLDL (density <1.006) was separated by ultracentrifugation. Then the apo CI, CIII, and B concentrations in VLDL were measured by enzyme-linked immunosorbent assay (ELISA)., Results: The apo CI, CIII, and B concentrations in VLDL were respectively 3-, 2-, and 2-fold higher, respectively, in diabetic patients with overt proteinuria than in controls. Hemodialysis patients with diabetic nephropathy had levels of apo CI, CIII, and B in VLDL that were 2.6-, 2.7- and 2-fold higher, respectively, than those in controls. Nondiabetic hemodialysis patients also had a 2.7-fold higher level of VLDL apo CIII, whereas VLDL apo CI and VLDL apo B were not significantly increased. VLDL apo CI was significantly correlated with VLDL apo B independently of VLDL apo CIII level., Conclusion: An increase of VLDL apo CIII is a prominent feature of dyslipidemia in CRF patients, regardless of whether they are diabetic or nondiabetic, whereas an increase of VLDL apo CI is more specific to diabetic nephropathy and is closely associated with an increase of VLDL particle numbers, a new risk factor for CHD.

To probe the role of protein conformation in the formation and kinetic stability of discoidal lipoproteins, thermal unfolding and refolding studies were carried out using model lipoproteins reconstituted from dimyristoylphosphatidylcholine (DMPC) and selected mutants of human apolipoprotein C-1 (apoC-1). Circular dichroism (CD) spectroscopy and electron microscopy show that the Q31P mutant, which has alpha-helical content in solution (33%) and on DMPC disks (67%) similar to that of the wild type (WT), forms disks of smaller diameter, = 13 nm, compared to 17 nm of the WT-DMPC disks. The L34P mutant, which is largely unfolded in solution, forms disks with alpha-helix content and diameter similar to those of the WT. The R23P mutant, which is fully unfolded in solution, forms disks that have similar diameter but reduced alpha-helix content (40%) compared to the WT-DMPC disks (65%). Remarkably, despite large variations in the alpha-helix content or the disk diameter among different mutant-DMPC complexes, the mutations have no significant effect on the unfolding rates or the Arrhenius activation energy of the disk denaturation, E(a) = 25-29 kcal/mol. This suggests that the kinetic stability of the discoidal complexes is dominated by the lipid-lipid rather than the protein-lipid interactions. In contrast to the heat denaturation, the lipoprotein reconstitution upon cooling monitored by CD and light scattering is significantly affected by mutations, with Q31P forming disks in the broadest and R23P in the narrowest temperature range. Our results suggest that the apolipoprotein helical structure in solution facilitates reconstitution of discoidal lipoproteins but has no significant effect on their kinetic stability.

Previous research has shown that polymorphisms of the apolipoproteins E ( APOE) and APOC1 represent genetic risk factors for dementia and for cognitive impairment in the elderly. The brain mechanisms by which these genetic variations affect behavior or clinical severity are poorly understood. We studied the effect of APOE and APOC1 genes on magnetic resonance imaging measures in a sample of 50 subjects with age-associated memory impairment. The APOE E4 allele was associated with reduced left hippocampal volumes and APOE*E3 status was associated with greater frontal lobe white matter volumes. However, no APOE effects were observed when analyses accounted for other potential confounding variables. The effects of APOC1 on hippocampal volumes appeared to be more robust than those of the APOE polymorphism. However, no modulatory effects on brain morphology outside the medial temporal lobe region were observed when demographic variables, clinical status, and other anatomical brain measurements were taken into consideration. Our results suggest that the role of the APOC1 polymorphism in brain morphology of the cognitively impaired elderly should be examined in further studies.

Apolipoproteins E and CI are the predominant components of triglyceride-rich lipoproteins. The genes are located in one gene cluster and both are polymorphic. Three allelic (epsilon2, epsilon3 and epsilon4) polymorphisms of the APOE gene influence plasma cholesterol levels. The distribution of these alleles differ between ethnic groups. PCR genotyping was used to determine the APOE and APOCI allele incidence in a representative group of 653 probands (302 men and 351 women) of Czech origin. The observed relative frequencies for the epsilon2, epsilon3 and epsilon4 alleles were 7.1 %, 82.0 % and 10.9 %, respectively, and are similar to other middle European populations. APO epsilon4 carriers have the highest and APO epsilon2 carriers the lowest levels of plasma total cholesterol (p<0.0001) and LDL cholesterol (p<0.0001). The frequency of the insertion (I) allele (HpaI restriction site present) of the APOCI polymorphism was 18.5 %. APOCI I/I homozygotes have the highest level of triglycerides (p<0.003). An almost complete linkage disequilibrium of the insertion allele of APOCI with the APOE alleles epsilon2 and epsilon4 has been detected and suggests that the deletion in the APOCI gene probably follows the deriving of all three APOE alleles on the APO epsilon3 allele background.

Apolipoprotein C-I (apoC-I) has been proposed to act primarily via interference with apoE-mediated lipoprotein uptake. To define actions of apoC-I that are independent of apoE, we crossed a moderately overexpressing human apoC-I transgenic, which possesses a minimal phenotype in the WT background, with the apoE-null mouse. Surprisingly, apoE-null/C-I mice showed much more severe hyperlipidemia than apoE-null littermates in both the fasting and non-fasting states, with an almost doubling of cholesterol, primarily in IDL+LDL, and a marked increase in triglycerides; 3-fold in females to 260 +/- 80 mg/dl and 14-fold in males to 1409 +/- 594 mg/dl. HDL lipids were not significantly altered but HDL were apoC-I-enriched and apoA-II-depleted. Production rates of VLDL triglyceride were unchanged as was the clearance of post-lipolysis remnant particles. Plasma post-heparin hepatic lipase and lipoprotein lipase levels were undiminished as was the in vitro hydrolysis of apoC-I transgenic VLDL. However, HDL from apoC-I transgenic mice had a marked inhibitory effect on hepatic lipase activity, as did purified apoC-I. LPL activity was minimally affected. Atherosclerosis assay revealed significantly increased atherosclerosis in apoE-null/C-I mice assessed via the en face assay. Inhibition of hepatic lipase may be an important mechanism of the decrease in lipoprotein clearance mediated by apoC-I.

Objectives: Recently, we reported that exaggerated postprandial triglyceridemia in normolipidemic patients with coronary artery disease is associated with enrichment of remnant lipoproteins with apolipoprotein C-I (apoC-I). In this study, the number and composition of chylomicron remnants and very low density lipoproteins (VLDLs) were examined in 30 asymptomatic normolipidemic 50-year-old men with and without early carotid atherosclerotic lesions., Methods and Results: Intima-media thickness of the far wall of the common carotid artery was determined by B-mode ultrasound. Triglyceride-rich lipoproteins were subfractionated by density gradient ultracentrifugation and separated into VLDL and chylomicron remnant fractions by immunoaffinity chromatography. The postprandial triglyceridemia and increase in triglyceride-rich lipoprotein particle number (ie, apolipoprotein B concentrations) were not exaggerated in men with early atherosclerosis. In contrast, their large (Svedberg flotation rate 60 to 400) and small (Svedberg flotation rate 20 to 60) chylomicron remnants and VLDL were greatly enriched with apoC-I, and their small chylomicron remnants and VLDL particles were relatively enriched with cholesterol. Moreover, the number of apoC-I molecules on small chylomicron remnants was strongly associated with the degree of atherosclerosis., Conclusions: Early asymptomatic atherosclerosis in normolipidemic men without exaggerated postprandial triglyceridemia is associated with the enrichment of postprandial chylomicron and VLDL particles with apoC-I. Therefore, it is conceivable that the apoC-I content of lipoprotein remnants may serve as an early marker of coronary artery disease risk.

Lipid-loaded macrophage "foam cells" accumulate in the subendothelial space during the development of fatty streaks and atherosclerotic lesions. To better understand the consequences of such lipid loading, murine peritoneal macrophages were isolated and incubated with ligands for two nuclear receptors, liver X receptor (LXR) and retinoic acid receptor (RXR). Analysis of the expressed mRNAs using microarray technology led to the identification of four highly induced genes that encode apolipoproteins E, C-I, C-IV, and C-II. Northern blot analysis confirmed that the mRNA levels of these four genes were induced 2-14-fold in response to natural or synthetic ligands for LXR and/or RXR. The induction of all four mRNAs was greatly attenuated in peritoneal macrophages derived from LXRalpha/beta null mice. The two LXR response elements located within the multienhancers ME.1 and ME.2 were shown to be essential for the induction of apoC-II promoter-reporter genes by ligands for LXR and/or RXR. Finally, immunohistochemical studies demonstrate that apoC-II protein co-localizes with macrophages within murine arterial lesions. Taken together, these studies demonstrate that activated LXR induces the expression of the apoE/C-I/C-IV/C-II gene cluster in both human and murine macrophages. These results suggest an alternative mechanism by which lipids are removed from macrophage foam cells.

Transgenic mice expressing human cholesteryl ester transfer protein (HuCETPTg mice) were crossed with apolipoprotein CI-knocked out (apoCI-KO) mice. Although total cholesterol levels tended to be reduced as the result of CETP expression in HuCETPTg heterozygotes compared with C57BL6 control mice (-13%, not significant), a more pronounced decrease (-28%, p < 0.05) was observed when human CETP was expressed in an apoCI-deficient background (HuCETPTg/apoCI-KO mice). Gel permeation chromatography analysis revealed a significant, 6.1-fold rise (p < 0.05) in the cholesteryl ester content of very low density lipoproteins in HuCETPTg/apoCI-KO mice compared with control mice, whereas the 2.7-fold increase in HuCETPTg mice did not reach the significance level in these experiments. Approximately 50% decreases in the cholesteryl ester content and cholesteryl ester to triglyceride ratio of high density lipoproteins (HDL) were observed in HuCETPTg/apoCI-KO mice compared with controls (p < 0.05 in both cases), with intermediate -20% changes in HuCETPTg mice. The cholesteryl ester depletion of HDL was accompanied with a significant reduction in their mean apparent diameter (8.68 +/- 0.04 nm in HuCETPTg/apoCI-KO mice versus 8.83 +/- 0.02 nm in control mice; p < 0.05), again with intermediate values in HuCETPTg mice (8.77 +/- 0.04 nm). In vitro purified apoCI was able to inhibit cholesteryl ester exchange when added to either total plasma or reconstituted HDL-free mixtures, and coincidently, the specific activity of CETP was significantly increased in the apoCI-deficient state (173 +/- 75 pmol/microg/h in HuCETPTg/apoCI-KO mice versus 72 +/- 19 pmol/microg/h in HuCETPTg, p < 0.05). Finally, HDL from apoCI-KO mice were shown to interact more readily with purified CETP than control HDL that differ only by their apoCI content. Overall, the present observations provide direct support for a potent specific inhibition of CETP by plasma apoCI in vivo.

Allele and haplotype frequencies for the T-93G, Hind III, and Pvu II variants of the lipoprotein lipase gene (LPL), and Hpa I and Ava II restriction site polymorphisms (RSP) of the APOE/C-I/C-II gene cluster were determined in 143 individuals from five Brazilian Indian tribes. These results were integrated with those previously reported for APOE. Marked interethnic variability occurs in these sites. A strong linkage disequilibrium was observed between the APOE and APOC-I loci (D' = 0.81; P < 0.00001). Linkage disequilibrium between the Hind III and Pvu II RSPs of the LPL gene was also observed (D' = 1; P < 0.001), but none of these RSPs were in linkage disequilibrium with the T-93G mutation. Considering both loci, heterozygosity was estimated as 0.45, but it was lower in the Xavante and Surui populations, in accordance with the historical and biodemographical data of these Amerindians. The results reported here may have implications for understanding interpopulation differences in lipid levels and coronary heart disease prevalences.

Thermal unfolding of discoidal complexes of apolipoprotein (apo) C-1 with dimyristoyl phosphatidylcholine (DMPC) reveals a novel mechanism of lipoprotein stabilization that is based on kinetics rather than thermodynamics. Far-UV CD melting curves recorded at several heating/cooling rates from 0.047 to 1.34 K/min show hysteresis and scan rate dependence characteristic of slow nonequilibrium transitions. At slow heating rates, the apoC-1 unfolding in the complexes starts just above 25 degrees C and has an apparent melting temperature T(m) approximately 48 +/- 1.5 degrees C, close to T(m) = 51 +/- 1.5 degrees C of free protein. Thus, DMPC binding may not substantially increase the low apparent thermodynamic stability of apoC-1, DeltaG(25 degrees C) < 2 kcal/mol. The scan rate dependence of T(m) and Arrhenius analysis of the kinetic data suggest an activation enthalpy E(a) = 25 +/- 5 kcal/mol that provides the major contribution to the free energy barrier for the protein unfolding on the disk, DeltaG > or = 17 kcal/mol. Consequently, apoC-1/DMPC disks are kinetically but not thermodynamically stable. To explore the origins of this kinetic stability, we utilized dynode voltage measured in CD experiments that shows temperature-dependent contribution from UV light scattering of apoC-1/DMPC complexes (d approximately 20 nm). Correlation of CD and dynode voltage melting curves recorded at 222 nm indicates close coupling between protein unfolding and an increase in the complex size and/or lamellar structure, suggesting that the enthalpic barrier arises from transient disruption of lipid packing interactions upon disk-to-vesicle fusion. We hypothesize that a kinetic mechanism may provide a general strategy for lipoprotein stabilization that facilitates complex stability and compositional variability in the absence of high packing specificity.

Since a deletion/insertion polymorphism in the promoter region of the apolipoprotein C-I (APOC1) gene has been reported to be associated with late-onset Alzheimer's disease (LOAD), we examined the hypothesis in a Korean population with 120 LOAD cases and 132 age-matched controls. The frequency of APOC1 insertion allele (H2) was significantly increased in LOAD than in controls, giving an odds ratio of 3.3 (95% CI 2.0-5.5, P<0.0001). Logistic regression analysis revealed that the interaction model between APOE epsilon4 and APOC1 H2 yielded larger odds ratio than other models including either APOE epsilon4 or APOC1 H2 alone. In addition, the association between APOC1 H2 and LOAD remained significant after adjustment of the effect of APOE epsilon4 (P=0.036). These results support previous observations that the APOC1 might be an additional susceptibility gene for LOAD.

Apolipoprotein (APO) C1 is a 6.6-kDa protein present in plasma and associated with lipoproteins. Using hyperinsulinemic-euglycemic clamp tests, we previously found that in APOC1 transgenic mice, the whole-body insulin-mediated glucose uptake is increased concomitant with a decreased fatty acid uptake. These latter results are confirmed in the present study, showing that APOC1 transgenic mice exhibit a 50% reduction in the uptake of the fatty acid analog 15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid in white adipose tissue stores. We next investigated whether APOC1 overexpression can modulate the initiation and/or development of obesity and insulin resistance. When crossbred on the genetically obese ob/ob background, APOC1 transgenic mice were fully protected from the development of obesity compared with ob/ob only mice, as reflected by a strong reduction in body weight (21 +/- 4 vs. 44 +/- 7 g), total adipose tissue stores (15 +/- 3 vs. 25 +/- 3% body wt), and average adipocyte size (7,689 +/- 624 vs. 15,295 +/- 1,289 microm(2)). Although less pronounced, APOC1 overexpression also reduced body weight on a wild-type background, solely due to a reduction in adipose tissue. Furthermore, despite elevated plasma free fatty acid and triglyceride levels, APOC1 overexpression significantly improved insulin sensitivity in ob/ob mice, as demonstrated by a strong reduction in plasma glucose and insulin levels, as well as a better performance in the glucose tolerance test. In conclusion, a marked reduction in the uptake of fatty acids into adipocytes may underlie the protection from obesity and insulin resistance in transgenic mice overexpressing human APOC1.

The H2 allele of apolipoprotein (apo) C-I is associated with Alzheimer's disease (AD). However, this association is potentially confounded by the linkage disequilibrium of H2 with the epsilon2 and epsilon4 alleles of apoE and of H1 with the epsilon3 allele. To establish plausibility for a direct role for apoC-I in AD, we compared apoC-I and apoE protein and mRNA levels in postmortem specimens of frontal cortex and hippocampus from AD patients with levels in nondemented controls. In H2-allelic individuals (usually also epsilon4 carriers), apoC-I mRNA levels were strikingly lower with AD (by 65%, P < 0.05), but apoC-I protein levels in AD were significantly higher (by 34%, P < 0.05). The opposite direction of the apoC-I mRNA and apoC-I protein level changes in AD in the epsilon4/H2 genotype may reflect decreased clearance of CNS lipoproteins associated with apoE4. In H1/H1 (usually epsilon3/epsilon3) individuals, both apoC-I protein and mRNA were lower in AD. ApoC-I protein levels in hippocampus were nearly twice those in frontal cortex. Immunohistochemistry of hippocampus revealed colocalization of apoC-I protein with the astrocytic marker GFAP. In addition, cultured human astrocytes expressed the mRNA for apoC-I. This study confirms apoC-I expression in the CNS and identifies astrocytes as the source of apoC-I. In addition, it has revealed differences in apoC-I expression based on site, genotype, and disease status that may reflect a role for apoC-I in the pathogenesis of AD.

A rabbit anti-serum to tree shrew apolipoprotein C-I (apo C-I) was used to screen an expression cDNA library constructed by us from tree shrew (TS) liver tissue. Two apo C-I cDNA clones were obtained. The longer one consists of 380 nucleotides, including 21 bp and 95 bp at the 5' and 3' end of the non-translated regions respectively, and a 264-bp fragment in an open reading frame encoding 88 amino acids prepropeptide which contains 26 amino acids of signal peptide and a mature protein (62 amino acids). Comparing the amino-acid sequence deduced from this cDNA with those of the published mammalian apo C-Is reveals that it shared some structural similarity with rat, mouse and dog apo C-I, but it had 5 more amino acids than that of human and baboon. The expression of apo C-I mRNA in 8 different tissues were also assayed with Northern blot. The results demonstrated that liver had the highest expression, intestine had much less expression and no expression in other tissues, which is much different from human and other species. This study has laid down a good foundation for further studying on the function and the stucture of tree shrew apo C-I gene.

Solution structure of lipid-free apolipoprotein C-1 (apoC-1, 6.6 kD) was analyzed by circular dichroism (CD) of 15 mutants containing single Pro or Ala substitutions in predicted alpha-helical regions. While the majority of Pro substitutions induce complete (L11P, L18P, R23P, I29P, M38P, W41P, T45P) or partial (G15P, L34P) helical unfolding, similar substitutions at other sites (A7P, Q31P, V49P, L53P) do not cause large changes in the secondary structure or stability. The results suggest that lipid-free apoC-1 is comprised of two dynamic helices that are stabilized by interhelical interactions and are connected by a short linker containing residues 30-33. We propose that the minimal folding unit in the lipid-free state of this and other exchangeable apolipoproteins comprises the helix-turn-helix motif formed of four 11-mer sequence repeats. Comparison of the helical content in lipid-free and lipid-bound apoC-1 suggests that lipid binding shifts the conformational equilibrium toward preexisting highly helical conformation. Remarkably, near-UV CD spectra of wild type and mutant apoC-1 are not significantly altered upon thermal or chemical unfolding and thus result from residual aromatic clustering that is retained in the unfolded state. Correlation of far- and near-UV CD of the mutant peptides suggests that the hydrophobic cluster containing W41 is essential for the helical stability and may form a helix nucleation site in apoC-1.

Apolipoprotein (apo)C-I and apoC-III are constituents of HDL and of triglyceride-rich lipoproteins that slow the clearance of triglyceride-rich lipoproteins by a variety of mechanisms. ApoC-I is an inhibitor of lipoprotein binding to the LDL receptor, LDL receptor-related protein, and VLDL receptor. It also is the major plasma inhibitor of cholesteryl ester transfer protein, and appears to interfere directly with fatty acid uptake. ApoC-III also interferes with lipoprotein particle clearance, but its principal role is as an inhibitor of lipolysis, both through the biochemical inhibition of lipoprotein lipase and by interfering with lipoprotein binding to the cell-surface glycosaminoglycan matrix where lipolytic enzymes and lipoprotein receptors reside. Variation in the expression of apoC-III has been credibly documented to have an important role in hypertriglyceridemia. Variation in the expression of apoC-I may also be important for hypertriglyceridemia under certain circumstances.

Mature human apolipoprotein C-I (apoC-I), comprising 57 amino acids, is the smallest member of the plasma apolipoprotein family. Amphipathic helical regions within apoC-I, common to this class of proteins, are mediators of lipid binding, a process that underlies the functional properties of apoC-I, including the capacity to activate the plasma enzyme LCAT, to disrupt apoE mediated receptor interactions and to inhibit cholesterol ester transfer protein. To examine apoC-I/phospholipid interactions, we have developed an expression system in Escherichia coli to obtain purified apoC-I with yields of approximately 4-5 mg per L of culture. The purified product has properties similar to plasma-derived apoC-I including self-association in the lipid-free state and induced alpha-helical content in the presence of egg-yolk phosphatidylcholine and dimyristoylglycerophosphocholine vesicles. We chose the short-chain phospholipid, dihexanoylglycerophosphocholine (Hex2Gro-PCho), to examine the interaction of apoC-I with submicellar phospholipid. Circular dichroism spectroscopy and cross-linking experiments show that apoC-I acquires helical content and remains self-associated at submicellar concentrations of Hex2Gro-PCho (4 mM). Sedimentation equilibrium studies of apoC-I at submicellar levels of Hex2Gro-PCho and analysis of the effects of apoC-I on the 1H NMR spectrum of Hex2Gro-PCho indicate micelle induction by apoC-I, and establish the capacity of apoC-I to assemble individual phospholipid molecules.

Aims/hypothesis: Insulin resistance for glucose metabolism is associated with hyperlipidaemia and high blood pressure. In this study we investigated the effect of primary hyperlipidaemia on basal and insulin-mediated glucose and on non-esterified fatty acid (NEFA) metabolism and mean arterial pressure in hyperlipidaemic transgenic mice overexpressing apolipoprotein C1 (APOC1). Previous studies have shown that APOC1 transgenic mice develop hyperlipidaemia primarily because of an impaired hepatic uptake of very low density lipoprotein (VLDL)., Methods: Basal and hyperinsulinaemic (6 mU.kg-1.min-1), euglycaemic (7 mmol/l) clamps with 3(-)3H-glucose or 9,10(-)3H-palmitic acid infusions and in situ freeze clamped tissue collection were carried out., Results: The APOC1 mice showed increased basal plasma cholesterol, triglyceride, NEFA and decreased glucose concentrations compared with wild-type mice (7.0 +/- 1.2 vs 1.6 +/- 0.1, 9.1 +/- 2.3 vs 0.6 +/- 0.1, 1.9 +/- 0.2 vs 0.9 +/- 0.1 and 7.0 +/- 1.0 vs 10.0 +/- 1.1 mmol/l, respectively, p < 0.05). Basal whole body glucose clearance was increased twofold in APOC1 mice compared with wild-type mice (18 +/- 2 vs 10 +/- 1 ml.kg-1.min-1, p < 0.05). Insulin-mediated whole body glucose uptake, glycolysis (generation of 3H2O) and glucose storage increased in APOC1 mice compared with wild-type mice (339 +/- 28 vs 200 +/- 11; 183 +/- 39 vs 128 +/- 17 and 156 +/- 44 vs 72 +/- 17 mumol.kg-1.min-1, p < 0.05, respectively), corresponding with a twofold to threefold increase in skeletal muscle glycogenesis and de novo lipogenesis from 3-(3)H-glucose in skeletal muscle and adipose tissue (p < 0.05). Basal whole body NEFA clearance was decreased threefold in APOC1 mice compared with wild-type mice (98 +/- 21 vs 314 +/- 88 ml.kg-1.min-1, p < 0.05). Insulin-mediated whole body NEFA uptake, NEFA oxidation (generation of 3H2O) and NEFA storage were lower in APOC1 mice than in wild-type mice (15 +/- 3 vs 33 +/- 6; 3 +/- 2 vs 11 +/- 4 and 12 +/- 2 vs 22 +/- 4 mumol.kg-1.min-1, p < 0.05) in the face of higher plasma NEFA concentrations (1.3 +/- 0.3 vs 0.5 +/- 0.1 mmol/l, p < 0.05), respectively. Mean arterial pressure and heart rate were similar in APOC1 vs wild-type mice (82 +/- 4 vs 85 +/- 3 mm Hg and 459 +/- 14 vs 484 +/- 11 beats.min-1)., Conclusions/interpretation: 1) Hyperlipidaemic APOC1 mice show reduced NEFA and increased glucose metabolism under both basal and insulin-mediated conditions, suggesting an intrinsic defect in NEFA metabolism. Primary hyperlipidaemia alone in APOC1 mice does not lead to insulin resistance for glucose metabolism and high blood pressure.

To examine the potential regulatory involvement of retroelements in the human genome, we screened the transcribed sequences of GenBank and expressed sequence tag data bases with long terminal repeat (LTR) elements derived from different human endogenous retroviruses. These screenings detected human transcripts containing LTRs belonging to the human endogenous retrovirus-E family fused to the apolipoprotein CI (apoC-I) and the endothelin B receptor (EBR) genes. However, both genes are known to have non-LTR (native) promoters. Initial reverse transcription-polymerase chain reaction experiments confirmed and authenticated the presence of transcripts from both the native and LTR promoters. Using a 5'-rapid amplification of cDNA ends protocol, we showed that the alternative transcripts of apoC-I and EBR are initiated and promoted by the LTRs. The LTR-apoC-I fusion and native apoC-I transcripts are present in many of the tissues tested. As expected, we found apoC-I preferentially expressed in liver, where about 15% of the transcripts are derived from the LTR promoter. Transient transfections suggest that the expression is not dependent on the LTR itself, but the presence of the LTR increases activity of the apoC-I promoter from both humans and baboons. The native EBR-driven transcripts were also detected in many tissues, whereas the LTR-driven transcripts appear limited to placenta. In contrast to the LTR of apoC-I, the EBR LTR promotes a significant proportion of the total EBR transcripts, and transient transfection results indicate that the LTR acts as a strong promoter and enhancer in a placental cell line. This investigation reports two examples where LTR sequences contribute to increased transcription of human genes and illustrates the impact of mobile elements on gene and genome evolution.

The aim of the present study was to identify the protein that accounts for the cholesteryl ester transfer protein (CETP)-inhibitory activity that is specifically associated with human plasma high density lipoproteins (HDL). To this end, human HDL apolipoproteins were fractionated by preparative polyacrylamide gradient gel electrophoresis, and 30 distinct protein fractions with molecular masses ranging from 80 down to 2 kDa were tested for their ability to inhibit CETP activity. One single apolipoprotein fraction was able to completely inhibit CETP activity. The N-terminal sequence of the 6-kDa protein inhibitor matched the N-terminal sequence of human apoC-I, the inhibition was completely blocked by specific anti-apolipoprotein C-I antibodies, and mass spectrometry analysis confirmed the identity of the isolated inhibitor with full-length human apoC-I. Pure apoC-I was able to abolish CETP activity in a concentration-dependent manner and with a high efficiency (IC(50) = 100 nmol/liter). The inhibitory potency of total delipidated HDL apolipoproteins completely disappeared after a treatment with anti-apolipoprotein C-I antibodies, and the apoC-I deprivation of native plasma HDL by immunoaffinity chromatography produced a mean 43% rise in cholesteryl ester transfer rates. The main localization of apoC-I in HDL and not in low density lipoprotein in normolipidemic plasma provides further support for the specific property of HDL in inhibiting CETP activity.

A 38-residue protein associated with cholesteryl ester transfer inhibition has been identified in baboons (Papio sp.). The cholesteryl ester transfer inhibitor protein (CETIP) corresponds to the N-terminus of baboon apoC-I. Relative to CETIP, baboon apoC-I is a weak inhibitor of baboon cholesteryl ester transferase (CET). To study the structural features responsible for CET inhibition, CETIP was synthesized by solid-phase methods. Using sodium dodecyl sulfate (SDS) to model the lipoprotein environment, the solution structure of CETIP was probed by optical and 1H NMR spectroscopy. Circular dichroism data show that the protein lacks a well-defined structure in water but, upon the addition of SDS, becomes helical (56%). A small blue shift of 8 nm was observed in the intrinsic tryptophan fluorescence of CETIP in the presence of saturating amounts of SDS, suggesting that tryptophan-23 is not buried deeply in the lipid environment. The helical nature of CETIP in the presence of SDS was confirmed by upfield 1Halpha secondary shifts and an average solution structure determined by distance geometry/simulated annealing calculations using 476 NOE-based distance restraints. The backbone (N-Calpha-C=O) root-mean-square deviation of an ensemble of 17 out of 25 calculated structures superimposed on the average structure was 1.06+0.30 A using residues V4-P35 and 0.51+/-0.17 A using residues A7-S32. Although the side-chain orientations fit the basic description of a class A amphipathic helix, both intramolecular salt bridge formation and "snorkeling" of basic side chains toward the polar face play minor, if any, roles in stabilizing the lipid-bound amphipathic structure. Conformational features of the calculated structures for CETIP are discussed relative to models of CETIP inhibition of cholesteryl ester transferase.

It is shown that apoC-III, but not other apoC proteins, components of very low density lipoproteins (apoC-I, apoC-II, apoC-III), reduced the rate of mitochondrial respiration in various metabolic states. This effect depended on the dose of apoprotein, type of oxidized substrate, and the presence of Ca ions in the incubation medium. ApoC-III completely blocked oxidative phosphorylation during oxidation of palmitoyl carnitine by mitochondria, while the respiration rate in metabolic state 4 remained unchanged.

We have previously shown that several multicopy gene families within the major histocompatibility complex (MHC) arose from a process of segmental duplication. It has also been observed that retroelements play a role in generating diversity within these duplicated segments. The objective of this study was to compare the genomic organization of a gene duplication within another multicopy gene family outside the MHC. Using new continuous genomic sequence encompassing the APOE-CII gene cluster, we show that APOCI and its pseudogene, APOCI', are contained within large duplicated segments which include sequences from the hepatic control region (HCR). Flanking Alu sequences are observed at both ends of the duplicated unit, suggesting a possible role in the integration of these segments. As observed previously within the MHC, the major differences between the segments are the insertion of sequences (approximately 200-1000 bp in length), consisting predominantly of Alu sequences. Ancestral retroelements also contribute to the generation of sequence diversity between the segments, especially within the 3' poly(A) tract of Alu sequences. The exonic and regulatory sequences of the APOCI and HCR loci show limited sequence diversity, with exon 3 being an exception. Finally, the typing of pre- and postduplication Alus from both segments indicates an estimated time of duplication of approximately 37 million years ago (mya), some time prior to the separation of Old and New World monkeys.

Background: Exaggerated postprandial triglyceridemia is common in normolipidemic patients with coronary artery disease (CAD). Alterations in the composition of triglyceride-rich lipoproteins (TRLs) are likely to underlie this metabolic disturbance. However, the composition of very-low-density lipoproteins (VLDLs), which are the most abundant postprandial TRLs, has never been defined in CAD patients., Methods and Results: We examined postprandial changes in the number and composition of VLDLs in middle-aged, normolipidemic CAD patients and control subjects. TRLs from 14 patients and 14 control subjects aged 45 to 55 years were subfractionated by density gradient ultracentrifugation into Svedberg flotation rate (Sf) fractions >400, 60 to 400, and 20 to 60. The VLDLs were separated from chylomicron remnants by immunoaffinity chromatography. In CAD patients, the postprandial concentrations of triglycerides and large (Sf 60 to 400) VLDL particles were elevated. In addition, their postprandial large VLDLs were enriched in apolipoprotein (apo) C-I and their postprandial small (Sf 20 to 60) VLDL remnants were enriched with apo C-I and cholesterol., Conclusions: Perturbed handling of postprandial triglycerides in normolipidemic CAD patients involves the accumulation of apo C-I-rich large VLDL particles and the generation of small, apo C-I- and cholesterol-rich VLDL remnants.

We have constructed a long-range restriction map spanning about 250 kb on bovine chromosome 18q24. Our results show that the apolipoprotein C2 (APOC2) gene is located about 25 kb from the APOE gene. Four putative CpG islands are also indicated in the map. Interestingly, a minisatellite located in the third intron of the human and mouse APOC2 genes was also found at identical position in the bovine gene and revealed high sequence identity comparing with the two corresponding sequences. By means of cosmid mapping, we further demonstrate that the APOE-APOC1-APOC2 gene cluster is evolutionary conserved in cattle.

Studies in humans on the in vivo metabolism of apolipoprotein (apo) Cs have been hampered by the highly complex nature of lipoprotein metabolism, which can be influenced by multiple genetic and environmental factors. In order to gain new insights into the function of the individual apoCs in lipoprotein metabolism, several laboratories have created mouse models lacking or overexpressing the respective APOC genes through the technologies of gene targeting and transgenesis. Until now, the only well-established in vivo metabolic function of apoC-I has been its inhibitory action on the uptake of very low-density lipoprotein (VLDL) via hepatic receptors, particularly the low-density lipoprotein (LDL) receptor-related protein. Consequently, the presence of apoC-I on the lipoprotein particle may prolong its residence time in the circulation and subsequently facilitate its conversion to LDL. ApoC-II, on the other hand, is a major activator of lipoprotein lipase, which is required for an efficient processing of triglyceride-rich lipoproteins in the circulation. However, an excess of apoC-II on the lipoprotein particle has been suggested to inhibit the lipoprotein-lipase-mediated hydrolysis of triglycerides. From studies with APOC3 transgenic and ApoC3-knockout mice, it appears that apoC-III inhibits the lipolysis of triglyceride-rich lipoproteins by hampering the interaction of these lipoproteins with the heparan sulfate proteoglycan-lipoprotein lipase complex. Subsequently, the poorly lipolyzed apoC-III-containing lipoprotein particles may accumulate in plasma because of their lower binding affinity towards hepatic receptors due to a change in lipid composition, particle size or the presence of apoC-III on the particle itself. From these data it can thus be concluded that all C apolipoproteins specifically modulate the metabolism of triglyceride-rich lipoproteins, which may contribute to the development of hyperlipidemia and other lipoprotein abnormalities in humans.

The joint distributions of phenotypes from the apolipoprotein E gene (APOE) and from a closely linked restriction site polymorphism at the apolipoprotein C1 locus (APOC1) were studied in population samples from Portugal and São Tomé e Príncipe (Gulf of Guinea), a former Portuguese colony that was originally populated by slaves imported from the African mainland. The frequencies of the APOE alleles (*2, *3, and *4) in Portugal and São Tomé fitted the ranges of variation generally observed in European and African populations, respectively. Haplotype analysis showed that in both populations the strength of linkage disequilibrium was highest for the APOE*2 allele and lowest for the APOE*4 allele, suggesting that the origin of the APOE alleles followed a 4-->3-->2 pathway and thus providing independent confirmation of the results from sequence homology studies with nonhuman primates. In accordance with global trends in the distribution of human genetic variation, the European sample from Portugal presented more intense linkage disequilibrium between APOE and APOC1 than the African sample from São Tomé where, despite the short 4-kb distance that separates the 2 loci, the level of association between the APOC1 alleles and APOE*4 was nonsignificant.

The high-resolution conformation of human apoC-I in complexes with sodium dodecyl sulfate (SDS) is presented. As estimated from CD data, apoC-I adopts 54% helical secondary structure when bound to SDS, which is similar to the helical content previously found with phospholipids. The NMR-derived conformation of apoC-I is composed of two amphipathic helices, residues 7-29 and 38-52, separated by a flexible linker. The N-terminal helix contains a mobile hinge involving residues 12-15. The hydrophobic side chains cluster on the nonpolar face of both helices, thus forming two discrete lipid-binding sites in the N-terminal helix and one in the C-terminal helix. As suggested by amide proton resonance line widths and deuterium exchange rates, the N-terminal helix is more flexible and may bind less tightly to the detergent than the C-terminal helix. The different mobility of both helices appears to be related to side-chain composition, rather than length of the amphipathic helix, and may play a role in the function of apoC-I as an activator of lecithin:cholesterol acyltransferase (LCAT). A model is suggested in which the C-terminal helix serves as a lipid anchor while the N-terminal helix may hinge off the lipid surface to make specific contacts with LCAT.