Sortilin enhances secretion of apolipoprotein(a) through effects on apolipoprotein B secretion and promotes uptake of lipoprotein(a)

Elevated plasma lipoprotein(a) (Lp(a)) is an independent, causal risk factor for atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Lp(a) is formed in or on hepatocytes from successive noncovalent and covalent interactions between apo(a) and apoB, although the subcellular location of these interactions and the nature of the apoB-containing particle involved remain unclear. Sortilin, encoded by the SORT1 gene, modulates apoB secretion and LDL clearance. We used a HepG2 cell model to study the secretion kinetics of apo(a) and apoB. Overexpression of sortilin increased apo(a) secretion, while siRNA-mediated knockdown of sortilin expression correspondingly decreased apo(a) secretion. Sortilin binds LDL but not apo(a) or Lp(a), indicating that its effect on apo(a) secretion is likely indirect. Indeed, the effect was dependent on the ability of apo(a) to interact noncovalently with apoB. Overexpression of sortilin enhanced internalization of Lp(a), but not apo(a), by HepG2 cells, although neither sortilin knockdown in these cells or Sort1 deficiency in mice impacted Lp(a) uptake. We found several missense mutations in SORT1 in patients with extremely high Lp(a) levels; sortilin containing some of these mutations was more effective at promoting apo(a) secretion than WT sortilin, though no differences were found with respect to Lp(a) internalization. Our observations suggest that sortilin could play a role in determining plasma Lp(a) levels and corroborate in vivo human kinetic studies which imply that secretion of apo(a) and apoB are coupled, likely within the hepatocyte.     

Acute suppression of apo B secretion by insulin occurs independently of MTP

Secretion of apolipoprotein (apo) B-containing lipoproteins by the liver depends mainly upon apo B availability and microsomal triglyceride transfer protein (MTP) activity and is subject to insulin regulation. Hepatic MTP mRNA expression is negatively regulated by insulin which correlates with inhibition of apo B secretion suggesting that insulin might suppress apo B secretion through an MTP-dependent mechanism. To investigate this possibility, we examined the acute effect of insulin on hepatic MTP expression and activity levels in vivo utilizing apobec-1^−/− mice. Insulin did not significantly alter hepatic MTP mRNA levels or lipid transfer activity 2 h following injection, but suppressed expression of genes important in gluconeogenesis. To study the specific role of MTP, we expressed human MTP (hMTP) in primary rat hepatocytes using adenoviral gene transfer. Increased expression of hMTP resulted in a 47.6 ± 17.9% increase in total apo B secreted. Incubation of hepatocytes with insulin suppressed apo B secretion by 50.1 ± 10.8% in cells over-expressing hMTP and by 53.0 ± 12.4% in control transfected hepatocytes. Results indicate that even under conditions of increased hepatic apo B secretion mediated by MTP, responsiveness of hepatocytes to insulin to suppress apo B secretion is maintained.     

Lipoprotein(a) and its position among other risk factors of atherosclerosis

Lipoprotein(a) [Lp(a)] comprises of an LDL particle and apolipoprotein(a) [apo(a)] and its elevated levels are considered a risk factor for atherosclerosis. The aim of our study was to find out whether elevated Lp(a) levels are associated with increased risk of atherosclerosis in patients with multiple other risk factors. We further tested the association of three polymorphisms of the apo(a) gene promoter with Lp(a) levels. No significant correlation was detected between Lp(a) levels and lipid and clinical parameters tested. The study demonstrated a significantly (p=0.0219) elevated Lp(a) level (mean 28+/-35 mg/dl, median 0.14) in patients with coronary heart disease (CHD). In a group with premature CHD the correlation was not significant anymore. There was a significant correlation between polymorphic loci of the promoter region of apo(a) gene and Lp(a) levels (+93C T, p=0.0166, STR, p<0.0001). Our study suggests that elevated Lp(a) level is an independent risk factor of CHD in carriers of other important CHD risk factors. Observed association of sequence variants of the promoter of apo(a) gene with Lp(a) levels is caused in part due to linkage to a restricted range of apo(a) gene length isoforms.     

Apolipoprotein CII (apo CII) gene expression defect in an individual with familial apo CII deficiency

We have examined the expression of the apolipoprotein CII (apo CII) gene in an individual with familial apo CII (apo CII) deficiency. Total RNA was prepared from this patient's liver tissue and analysed in Slot Blot and Northern Blot experiments using a cloned apo CII cDNA as a probe. In this patient, there is at least a four-fold decrease in the level of apo CII mRNA, when compared to liver tissue from a control individual. The residual apo CII mRNA detected in this patient is of normal length. These results suggest that the failure to detect apo CII protein in this patient's serum is not due to a failure to transcribe or process apo CII mRNA, but probably to a defect in the translation of the apo CII message. This defect results in partial degradation of the apo CII message leading to the much reduced levels which we have observed.     

Isolation and partial characterization of apolipoprotein (a) from human lipoprotein (a)

A procedure was developed for the dissociation of apolipoprotein (a) (apo (a)) from pure human lipoprotein (a) (Lp(a)) prepared by density gradient ultracentrifugation and gel filtration. Lp(a) was ultracentrifuged through a layer of saline which was adjusted to a density of 1.182 g/mL and contained 30 mM dithiothreitol (50 mM) and phenylmethylsulfonyl fluoride (1.25 mM). Following centrifugation, the lipid and apolipoprotein B (apo B) were recovered as a lipoprotein (Lp(a) B) in the supernatant fraction, while the apo (a) was recovered as a lipid-poor protein pellet. An investigation of the supernatant lipoprotein by electron microscopy and compositional analysis revealed that it was similar in size and composition to low density lipoprotein (LDL) isolated from the same density range and contained apo B100 with an amino acid and carbohydrate composition which was similar to apo B from LDL. Estimates of the apparent molecular weight of the apo (a) varied amongst individuals but was always greater than apo B100 (congruent to 450,000). The amino acid composition of apo (a), which was very distinct from apo B, was characterized by a higher content of serine, threonine, proline, and tyrosine, but lower amounts of isoleucine, phenylalanine, and lysine when compared with apo B of Lp(a) or LDL. The apo (a) contained a much higher proportion of carbohydrate, in particular N-acetylgalactosamine, galactose, and N-acetylneuraminic acid (which were three- to six-fold higher) than the apo B of Lp(a). It is concluded that apo (a) is distinct from other apolipoproteins owing to its low avidity for lipid and the nature of the interaction with apo B. Lp(a) consists of an LDL-like particle with a carbohydrate-rich apo (a) attached to the surface of apo B.     

Variation in Lp(a) levels and apo(a) isoform frequencies in five baboon subspecies

Elevated levels of lipoprotein (a) [Lp(a)] are positively correlated with risk of cardiovascular disease and are thought to be a function of allelic variation in apo(a), the unique protein component of Lp(a). In this article we examine subspecies variation in Lp(a) levels and apo(a) isoforms in the baboon. Breeding populations of the five subspecies (Papio hamadryas hamadryas, P.h. cynocephalus, P.h. ursinus, P.h. papio, and P.h. anubis) of common long-tailed baboons are maintained at the Southwest Foundation for Biomedical Research. Serum samples were obtained from at least 20 unrelated animals of each subspecies. Twelve different size isoforms (including the null) of apo(a) were identified across the five subspecies. These isoforms act as alleles; a maximum likelihood method was used to obtain the allele frequencies. Significant differences in apo(a) isoform frequencies were found between subspecies (chi 2(44) = 163.10, p less than 0.0001). Quantitative levels of Lp(a) also differed among subspecies. We evaluated the correlation between genetic distances calculated using the quantitative Lp(a) levels and the apo(a) isoform data. Observed genetic relationships among the subspecies are consistent with the present-day geographic distribution and information from other marker protein systems. The findings indicate that the marker apo(a) may have great utility in both evolutionary and biomedical studies.     

6-Aminohexanoic acid as a chemical chaperone for apolipoprotein(a)

Apolipoprotein (a) (apo(a)) is a component of the atherogenic lipoprotein, Lp(a). The efficiency with which apo(a) escapes the endoplasmic reticulum (ER) and is secreted by the liver is a major determinant of plasma Lp(a) levels. Apo(a) contains a series of domains homologous to plasminogen kringle (K) 4, each of which possesses a potential lysine-binding site. By using primary mouse hepatocytes expressing a 17K4 human apo(a) protein, we found that high concentrations (25-200 mM) of the lysine analog, 6-aminohexanoic acid (6AHA), increased apo(a) secretion 8-14-fold. This was accompanied by a decrease in apo(a) presecretory degradation. 6AHA inhibited accumulation of apo(a) in the ER induced by the proteasome inhibitor, lactacystin. Thus, 6AHA appeared to inhibit degradation by increasing apo(a) export from the ER. Significantly, 6AHA overcame the block in apo(a) secretion induced by the ER glucosidase inhibitor, castanospermine. 6AHA may therefore circumvent the requirement for calnexin and calreticulin interaction in apo(a) secretion. Sucrose gradients and a gel-based folding assay were unable to detect any influence of 6AHA on apo(a) folding. However, non-covalent or small, disulfide-dependent changes in apo(a) conformation would not be detected in these assays. Proline also increased the efficiency of apo(a) secretion. We propose that 6AHA and proline can act as chemical chaperones for apo(a).     

Effects of epidermal growth factor on apo B mRNA levels and apo B accumulation in the media of primate hepatocytes in culture.

EGF has been shown to augment albumin and apolipoprotein A-I secretion by cynomolgus monkey hepatocytes in primary culture without stimulating cell division. This study was undertaken to determine what effect EGF had on apo B secretion by those hepatocytes. The results indicate that EGF (3 nM final concentration) severely inhibits the rate at which apo B accumulates in the culture medium of primate hepatocytes. That effect was evident within 48 hours of treatment, and by 72 hours the rate that apo B accumulated was less than half that of cells treated with a hormone-free medium. However, the apo B mRNA levels in the EGF-treated cells were more than double those of hepatocytes given the hormone-free medium. These data indicate that EGF has a potent effect on the rate at which apo B accumulates in the culture medium of primate hepatocytes and that the effect is independent of apo B gene expression.     

Production of lipoprotein(a) by primary baboon hepatocytes

Primary baboon hepatocytes were cultured in a serum-free medium formulation that permitted the analysis of lipoprotein(a) (Lp(a] production by the cells. The hepatocytes were determined to synthesize Lp(a) on the basis of the following observations: (1) the culture medium reacted in an ELISA designed for detection of baboon Lp(a) in serum samples; (2) the Lp(a)-specific protein, apo(a), was detected in the culture medium by immunoblotting techniques; (3) the unique protein structure of Lp(a) was demonstrated (i.e., association of apo(a) with apoB via interchain disulfide bonds to form apoLp(a]; and (4) the Lp(a) proteins occurred in the medium at a density of about 1.05 g/ml when subjected to density gradient ultracentrifugation. De novo synthesis of Lp(a) by cultured hepatocytes was demonstrated by incorporation of [35S]cysteine. Lp(a) was produced by the hepatocytes throughout a 20 day culture period. Finally, apo(a) isoform patterns in the hepatocyte culture medium and the hepatocyte donors' serum were indistinguishable.