Inhibition of N-linked glycosylation results in retention of intracellular apo[a] in hepatoma cells, although nonglycosylated and immature forms of apolipoprotein[a] are competent to associate with apolipoprotein B-100 in vitro

Apolipoprotein[a] (apo[a]) is a highly polymorphic glycoprotein that forms a covalent complex with apolipoprotein B-100 (apoB-100), producing a lipoprotein species referred to as lipoprotein[a] (Lp[a]). We have studied the effects of alterations in glycosylation of apo[a] on its intracellular processing and secretion as well as its ability to associate with low density lipoprotein (LDL) apoB-100. HepG2 cells transfected with a 6 kringle IV (6 K-IV) apo[a] minigene were treated with tunicamycin, an inhibitor of N-linked glycosylation, which eliminated apo[a]-B-100 complexes from the media. Tunicamycin treatment also reduced secretion of the 6 K-IV apo[a] protein from transfected McA-RH7777 cells by approximately 50%, but completely eliminated secretion of apo[a] species containing 9 and 17 K-IV repeats. Mixing experiments, performed with radiolabeled media (+/-tunicamycin) from transfected McA-RH7777 cells, demonstrated no alteration in the extent of association of apo[a] with human LDL. Similar mixing experiments using culture media from glycosylation-defective mutant chinese hamster ovary (CHO) cells transfected with the same apo[a] minigene showed identical results. Apo[a] secretion was demonstrated in all mutant cell lines in the absence of either N- or O-linked (or both) glycosylation. The mechanisms underlying the reduced secretion of apo[a] from transfected hepatoma cells were examined by pulse-chase radiolabeling and apo[a] immunoprecipitation. Tunicamycin treatment altered the efficiency of precursor apo[a] processing from the ER by increasing its ER retention time. The increased accumulation of precursor apo[a] in the ER was associated with alterations in the kinetics of association with two resident endoplasmic reticulum (ER) chaperone proteins, calnexin and BiP. These findings suggest that the glycosylation state and size of apo[a] appear to play a role in regulating its efficient exit from the endoplasmic reticulum. However, neither N- nor O-linked glycosylation of apo[a] exerts a major regulatory role in its covalent association with apoB-100.     

Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome

Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.     

Neomycin inhibits secretion of apolipoprotein[a] by increasing retention on the hepatocyte cell surface

Neomycin therapy reduces plasma levels of low density lipoprotein and lipoprotein[a] (Lp[a]). To determine whether neomycin directly alters the biogenesis of Lp[a], we have examined the effect of neomycin on apolipoprotein[a] (apo[a]) synthesis and secretion in primary cultures of baboon hepatocytes. Using this system, we have previously shown that apo[a] is synthesized as a lower molecular weight precursor that upon maturation becomes associated with the cell surface before release into the culture medium. Treatment of hepatocytes with 10 mM neomycin reduced levels of apo[a] in the culture medium by as much as 12-fold. Although a portion of the reduced secretion could be accounted for by a reduction in total protein synthesis, the greatest effect of neomycin on apo[a] secretion was to decrease the release of mature apo[a] from the hepatocyte cell surface into the culture medium. Treatment of hepatocyte cultures with trypsin confirmed that mature apo[a] in neomycin-treated cells was still transported to the cell surface. Examination of related antibiotics demonstrated that inhibition of apo[a] secretion is a general property shared by the deoxystreptamine antibiotics. The mechanism by which neomycin affects the apo[a]-cell surface interaction is not known, but neomycin is known to perturb cell surface membranes, inhibit the interaction of some ligands with their cell surface receptors, and inhibit the metabolism of phosphatidylinositol 4,5 biphosphate. These studies suggest that cell surface association of apo[a] may play a role in Lp[a] biogenesis in vivo.     

CD4 glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic beta subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E1 or (ii) following expression of a mutant form of ubiquitin (Lys48 mutated to Arg48) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.     

Effects of TCDD on Ah receptor, ARNT, EGF, and TGF-alpha expression in embryonic mouse urinary tract

Lipoprotein(a) [Lp(a)] biogenesis was examined in primary cultures of hepatocytes isolated from mice transgenic for both human apolipoprotein(a) [apo(a)] and human apoB. Steady-state and pulse-chase labeling experiments demonstrated that newly synthesized human apo(a) had a prolonged residence time (approximately 60 min) in the endoplasmic reticulum (ER) before maturation and secretion. Apo(a) was inefficiently secreted by the hepatocytes and a large portion of the protein was retained and degraded intracellularly. Apo(a) exhibited a prolonged and complex folding pathway in the ER, which included incorporation of apo(a) into high molecular weight, disulfide-linked aggregates. These folding characteristics could account for long ER residence time and inefficient secretion of apo(a). Mature apo(a) bound via its kringle domains to the hepatocyte cell surface before appearing in the culture medium. Apo(a) could be released from the cell surface by apoB-containing lipoproteins. These studies are consistent with a model in which the efficiency of post-translational processing of apo(a) strongly influences human plasma Lp(a) levels, and suggest that cell surface assembly may be one pathway of human Lp(a) production in vivo. Transgenic mouse hepatocytes thus provide a valuable model system with which to study factors regulating human Lp(a) biogenesis.     

Ubiquitination is required for the retro-translocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome

In the endoplasmic reticulum (ER), an efficient "quality control system" operates to ensure that mutated and incorrectly folded proteins are selectively degraded. We are studying ER-associated degradation using a truncated variant of the rough ER-specific type I transmembrane glycoprotein, ribophorin I. The truncated polypeptide (RI332) consists of only the 332 amino-terminal amino acids of the protein corresponding to most of its luminal domain and, in contrast to the long-lived endogenous ribophorin I, is rapidly degraded. Here we show that the ubiquitin-proteasome pathway is involved in the destruction of the truncated ribophorin I. Thus, when RI332 that itself appears to be a substrate for ubiquitination was expressed in a mutant hamster cell line harboring a temperature-sensitive mutation in the ubiquitin-activating enzyme E1 affecting ubiquitin-dependent proteolysis, the protein is dramatically stabilized at the restrictive temperature. Moreover, inhibitors of proteasome function effectively block the degradation of RI332. Cell fractionation experiments indicate that RI332 accumulates in the cytosol when degradation is prevented by proteasome inhibitors but remains associated with the lumen of the ER under ubiquitination-deficient conditions, suggesting that the release of the protein into the cytosol is ubiquitination-dependent. Accordingly, when ubiquitination is impaired, a considerable amount of RI332 binds to the ER chaperone calnexin and to the Sec61 complex that could effect retro-translocation of the polypeptide to the cytosol. Before proteolysis of RI332, its N-linked oligosaccharide is cleaved in two distinct steps, the first of which might occur when the protein is still associated with the ER, as the trimmed glycoprotein intermediate efficiently interacts with calnexin and Sec61. From our data we conclude that the steps that lead a newly synthesized luminal ER glycoprotein to degradation by the proteasome are tightly coupled and that especially ubiquitination plays a crucial role in the retro-translocation of the substrate protein for proteolysis to the cytosol.     

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Troglitazone is a new oral hypoglycemic agent that reduces insulin resistance in non-insulin-dependent diabetes mellitus (NIDDM). However, this agent increases serum lipoprotein(a) [Lp(a)], which is known as an atherogenic lipoprotein. The relationships between the response of Lp(a) to troglitazone and the apolipoprotein(a) [apo(a)] phenotype were investigated in this study. Nineteen NIDDM patients were treated with troglitazone for 4 weeks. Lp(a) increased significantly from 20.1+/-16.5 mg/dL to 44.1+/-31.9 mg/dL (P<.001) in all study patients. Lp(a) increased from 25.7+/-34.2 mg/dL to 50.1+/-38.7 mg/dL (P = .03) in patients with smaller apo(a) phenotypes (S1S4 to S2S4). Lp(a) also increased from 17.5+/-12.0 mg/dL to 41.3+/-29.6 mg/dL (P<.01) in patients with larger apo(a) phenotypes (S3 to S4). Therefore, the increase of Lp(a) by troglitazone may be independent of the apo(a) phenotype.     

Gemfibrozil significantly lowers cynomolgus monkey plasma lipoprotein[a]-protein and liver apolipoprotein[a] mRNA levels

Eight male cynomolgus monkeys (Macaca fascicularis) on a normal chow diet were orally administered gemfibrozil daily using a weekly rising dose protocol for 3 weeks (50, 125, and 200 mg/kg per day). At these drug doses, Lp[a] levels were reduced: 83.7% +/- 3.2 (SEM), (P < 0.024); 63.7% +/- 4.1 (P < 0.013); and 36.2% +/- 1.1 (P < 0.002), respectively, of pretreatment values. Lp[a] reduction was directly related to blood gemfibrozil concentration (range 36-428 microM, r = 0.969) and occurred without concomitant changes in apolipoprotein B. Three weeks posttreatment Lp[a] levels returned to pretreatment values. A specific ribonuclease protection assay demonstrated that liver apolipoprotein[a] (apo[a]) mRNA expression was decreased in all animals to an average of 19.1% +/- 3.0 (P < 0.0026), of pretreatment values after the 200 mg/kg treatment, whereas, albumin, apolipoprotein A-I, apolipoprotein E, and glyceraldehyde-3-phosphate dehydrogenase mRNAs were unchanged. Lp[a] levels were unaffected by gemfibrozil in HepG2 cells permanently transfected with an apo[a] 10-kringle cDNA construct containing partial 5'- and 3'-untranslated sequences and under control of a constitutive CMV promoter. However, both Lp[a] and apo[a] mRNA in primary cynomolgus monkey hepatocytes were coordinately lowered in a dose-dependent fashion by gemfibrozil. Thus, Lp[a] can be regulated by gemfibrozil at the level of apo[a] mRNA expression.     

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The effects of lipoproteins on ion channel-mediated catecholamine secretion were investigated in cultured bovine adrenal medullary cells. Low density lipoprotein (LDL: 20-80 mg/dl) and lipoprotein(a) [Lp(a); 10-80 mg/dl] inhibited catecholamine secretion induced by carbachol, an activator of nicotinic acetylcholine receptor-ion channels. LDL and Lp(a) suppressed carbachol-induced 22Na+ influx as well as 45Ca2+ influx in a concentration-dependent manner similar to that of catecholamine secretion. The inhibition of catecholamine secretion by Lp(a) was not overcome by increasing the concentration of carbachol. On the other hand, high density lipoprotein (HDL; < 150 mg/dl) had no effect on 22Na+ influx, 45Ca2+ influx, and catecholamine secretion. Like LDL and Lp(a), a synthetic peptide homologous to human plasma apolipoprotein B (apoB), apoB fragment(3358-3372)-amide (3-60 microM), attenuated 22Na+ influx, 45Ca2+ influx, and catecholamine secretion caused by carbachol. The apoB fragment also suppressed 22Na+ influx induced by veratridine (an activator of voltage-dependent Na+ channels) and 45Ca2+ influx induced by 56 mM K+ (an indirect activator of voltage-dependent Ca2+ channels). These findings suggest that atherogenic lipoproteins such as LDL and Lp(a) suppress catecholamine secretion by interfering with Na+ influx through nicotinic acetylcholine receptor-ion channels, in which apoB, a structural component common to both LDL and Lp(a), plays an important role. The inhibition by atherogenic lipoproteins of catecholamine secretion may influence the progression of atherosclerosis induced by these lipoproteins.     

Elevated lipoprotein (a) levels in primary nephrotic syndrome

Elevated plasma levels of total cholesterol and increase in the hepatic synthesis of some apo B-containing lipoproteins have been noted in the nephrotic syndrome. Apoprotein (a), the apolipoprotein distinguishing lipoprotein (a) [Lp(a)] from low-density lipoprotein, is equally of hepatic origin, and Lp(a) recently has been shown to possess both atherogenic and thrombogenic activities. However, little is known of Lp(a) levels in nephrotic patients. We measured plasma Lp(a) concentrations in 11 patients with primary nephrotic syndrome in the absence of hematuria, hypertension, and renal insufficiency. Histologic lesions were minimal-change disease in five cases, membranous glomerulopathy in four cases, and focal and segmental glomerulosclerosis in two cases. Mean levels of Lp(a) (98 +/- 92 mg/dL [mean +/- SD]) were markedly elevated in the nephrotic patients as compared with the controls (14 +/- 13 mg/dL). No correlation was noted between plasma Lp(a) and proteinuria, albuminemia, total cholesterolemia, low-density lipoprotein cholesterol, apoprotein B100, or plasminogen. Furthermore, there was no correlation between Lp(a) levels and apoprotein (a) isoform size. In four patients, the level of Lp(a) decreased approximately fourfold after remission of the nephrotic syndrome under corticosteroid treatment. Our observation that Lp(a) levels are elevated in the nephrotic syndrome is consistent with the hypothesis that these patients may be at an increased risk of cardiovascular and thrombotic complications.     

Apolipoprotein(a) induces monocyte chemotactic activity in human vascular endothelial cells

BACKGROUND: Elevated levels of lipoprotein(a) [Lp(a)] are associated with premature atherosclerosis; however, the mechanisms are not known. Recruitment of monocytes to the blood vessel wall is an early event in atherogenesis. METHODS AND RESULTS: This study has found that unoxidized Lp(a) induced human umbilical vein endothelial cells (HUVECs) to secrete monocyte chemotactic activity (MCA), whereas LDL under the same conditions did not. In the absence of HUVECs, Lp(a) had no direct MCA. Endotoxin was shown not to be responsible for the induction of MCA. Actinomycin D and cycloheximide inhibited the HUVEC response to Lp(a), indicating that protein and RNA synthesis were required. The apolipoprotein(a) [apo(a)] portion of Lp(a) was identified as the structural component of Lp(a) responsible for inducing MCA. Lp(a) and apo(a) also stimulated human coronary artery endothelial cells to produce MCA. Granulocyte-monocyte colony-stimulating factor (GM-CSF) antigen was not detected in the Lp(a)-conditioned medium, nor was monocyte chemoattractant protein-1 mRNA induced in HUVECs by Lp(a). CONCLUSIONS: These findings suggest that Lp(a) may be involved in the recruitment of monocytes to the vessel wall and provide a novel mechanism for the participation of Lp(a) in the atherogenic process.     

Lipoprotein Lp(a): effects of allelic variation at the LPA locus

Concentrations of the lipoprotein Lp(a) vary widely in healthy individuals, but in many studies, high concentrations are strongly associated with cardiovascular disease. On the basis of lipid and protein composition, Lp(a) is a variant of the atherogenic low-density lipoprotein but differs in possessing the unique apolipoprotein(a) [apo(a)]. Lp(a) concentrations are controlled at the level of biosynthesis of the apo(a) protein, which is encoded by the LPA locus, and allelic differences at LPA are responsible for the bulk of variation in Lp(a) phenotype. In this article we describe several aspects of allelic variation at LPA reported in studies of human and baboons, including (1) polymorphisms for protein size, (2) families of alleles having distinct relationships between apo(a) size and Lp(a) concentration, (3) sequence polymorphisms, (4) a group of alleles whose protein products have a multibanded phenotype, and (5) allelic diversity of null phenotype alleles (whose protein products are not detected in the plasma). The data make clear that no single aspect of allelic variation at LPA is sufficient to fully explain the genetic control of Lp(a).     

Effects of apoE gene polymorphism on Lp(a) concentrations depend on the size of apo(a): a study of 466 white men

Polymorphisms in the genes for the low-density lipoprotein (LDL) receptor ligands, apolipoprotein E (apoE), and apolipoprotein B (apoB) are associated with variation in plasma levels of LDL cholesterol. Lp(a) lipoprotein(a) [Lp(a)] is LDL in which apoB is attached to a glycoprotein called apolipoprotein(a) [apo(a)]. Apo(a) has several genetically determined isoforms differing in molecular weight, which are inversely correlated with Lp(a) concentrations in blood. The interaction of apo(a) with triglyceride-rich lipoproteins differs with the size of apo(a), and therefore the effects of apoE gene polymorphism on Lp(a) levels could also depend on apo(a) size. We have investigated the possible effect of genetic variation in the apoE and apoB genes on plasma Lp(a) concentrations in 466 white men with different apo(a) phenotypes. Overall there was no significant association between the common apoE polymorphism and Lp(a), but in the subgroup with apo(a)-S4, concentrations of Lp(a) differed significantly among the apoE genotypes (P = 0.05). Lp(a) was highest in the apoE genotypes epsilon 2 epsilon 3 and epsilon 3 epsilon 3 and lowest in genotype epsilon 3 epsilon 4, and the apoE polymorphism was estimated to account for about 2.4% of the variation in Lp(a). In contrast, in the subgroup with apo(a)-S2 Lp(a) was significantly lower (P = 0.04) in apoE genotype epsilon 2 epsilon 3 than in genotype epsilon 3 epsilon 3. Lp(a) concentrations did not differ among the XbaI (P = 0.65) or SP 24/27 (P = 0.26) polymorphisms of the apoB gene. The expected effects of both apoE and apoB polymorphism on LDL levels were significant in the whole population sample and in subjects with large-sized apo(a) isoforms (P < 0.01), whereas no effect was seen in those with low molecular weight apo(a) isoforms. We conclude that the influence of apoE genotypes on Lp(a) concentrations depends on the size of the apo(a) molecule in Lp(a), possibly because both apo(a)-S4 and apoE4 have high affinity for triglyceride-rich lipoproteins and may be taken up and degraded rapidly by remnant receptors.     

Intracellular metabolism of human apolipoprotein(a) in stably transfected Hep G2 cells

Lipoprotein(a) [Lp(a)] consists of LDL and the glycoprotein apolipoprotein(a) [apo(a)], which are covalently linked via a single disulfide bridge. The formation of Lp(a) occurs extracellularly, but an intracellular assembly in human liver cells has also been claimed. The human apo(a) gene locus is highly polymorphic due to a variable number of tandemly arranged kringle IV repeats. The size of apo(a) isoforms correlates inversely with Lp(a) plasma concentrations, which is believed to reflect different synthesis rates. To examine this association at the cellular level, we analyzed the subcellular localization and fate of apo(a) in stably transfected HepG2 cells. Our results demonstrate that apo(a) is synthesized as a precursor with a lower molecular mass which is processed into the mature, secreted form. The retention times of the precursor in the ER positively correlated with the sizes of apo(a) isoforms. The mature form was observed intracellularly at low levels and only in the Golgi apparatus. No apo(a) was found to be associated with the plasma membrane. Under temperature-blocking conditions, we did not detect any apo(a)/apoB-100 complexes within cells. This finding was confirmed in HepG2 cells transiently expressing KDEL-tagged apo(a). The precursor and the mature forms of apo(a) were found in the ER and Golgi fractions, respectively, also in human liver tissue. From our data, we conclude that in HepG2 cells the apo(a) precursor, dependent on the apo(a) isoform, is retained in the ER for a prolonged period of time, possibly due to an extensive maturation process of this large protein. The assembly of Lp(a) takes place exclusively extracellularly following the separate secretion of apo(a) and apoB.     

Proparathyroid hormone-related protein is associated with the chaperone protein BiP and undergoes proteasome-mediated degradation

Parathyroid hormone-related peptide (PTHrP) is an important causal factor for hypercalcemia associated with malignancy. In addition to the endocrine functions attributed to secretory forms of the peptide, PTHrP also plays a local role as a mediator of cellular growth and differentiation presumably at least in part through intracellular pathways. In studying the post-translational regulation of PTHrP, we observed that PTHrP was conjugated to multiple ubiquitin moieties. We report here that the proteasome is responsible for the degradation of the endoplasmic reticulum-associated precursor, pro-PTHrP. Cells expressing prepro-PTHrP and exposed to lactacystin accumulate pro-PTHrP assessed by anti-pro specific antibodies. Brefeldin A-treated cells also accumulate pro-PTHrP suggesting that degradation does not occur in the endoplasmic reticulum (ER) lumen. Subcellular fractionation of both lactacystin and brefeldin A-treated cells indicated that accumulated pro-PTHrP resides in microsomal fractions with a portion of the protein exposed to the cytosolic side of the ER membrane as assessed by protease protection experiments. Immunoprecipitation and Western blot analysis identified pro-PTHrP in association with the ER molecular chaperone protein BiP. We conclude that pro-PTHrP from the ER can gain access to the cytoplasmic side of the ER membrane where it can undergo ubiquitination and degradation by the proteasome.     

Lipoprotein(a) concentration and molecular weight of apolipoprotein(a) in patients with cerebrovascular disease and diabetes mellitus

Plasma lipoprotein(a) [Lp(a)] concentrations are genetically determined, and hyper-Lp(a)-emia is an independent risk factor for atherosclerosis and thrombosis. To study the implications of Lp(a) in cerebrovascular disease (CVD) and diabetes mellitus (DM), we examined plasma Lp(a) levels and molecular weights of apolipoprotein(a) [apo(a)] in 118 patients with CVD, and 125 cases with DM. Although mean Lp(a) concentrations were higher in those cases with atherothrombotic brain infarction than in those with brain hemorrhage and lacunar infarction, the difference was not statistically significant. Lp(a) levels were significantly higher in the DM cases treated with insulin and in those treated with oral hypoglycemic agents than in those on diet therapy alone, suggesting that insulin and oral agents modulate apo(a) expression. Lp(a) concentrations correlated significantly with the low-molecular-weight isoforms of apo(a) in all CVD and DM groups.     

Dramatically decreased high density lipoprotein cholesterol, increased remnant clearance, and insulin hypersensitivity in apolipoprotein A-II knockout mice suggest a complex role for apolipoprotein A-II in atherosclerosis susceptibility

Apolipoprotein (apo) A-II is the second most abundant apolipoprotein in high density lipoprotein (HDL). To study its role in lipoprotein metabolism and atherosclerosis susceptibility, apo A-II knockout mice were created. Homozygous knockout mice had 67% and 52% reductions in HDL cholesterol levels in the fasted and fed states, respectively, and HDL particle size was reduced. Metabolic turnover studies revealed the HDL decrease to be due to both decreased HDL cholesterol ester and apo A-I transport rate and increased HDL cholesterol ester and apo A-I fractional catabolic rate. The apo A-II deficiency trait was bred onto the atherosclerosis-prone apo E-deficient background, which resulted in a surprising 66% decrease in cholesterol levels due primarily to decreased atherogenic lipoprotein remnant particles. Metabolic turnover studies indicated increased remnant clearance in the absence of apo A-II. Finally, apo A-II deficiency was associated with lower free fatty acid, glucose, and insulin levels, suggesting an insulin hypersensitivity state. In summary, apo A-II plays a complex role in lipoprotein metabolism, with some antiatherogenic properties such as the maintenance of a stable HDL pool, and other proatherogenic properties such as decreasing clearance of atherogenic lipoprotein remnants and promotion of insulin resistance.