Lepodisiran, an Extended-Duration Short Interfering RNA Targeting Lipoprotein(a): A Randomized Dose-Ascending Clinical Trial.

Importance: Epidemiological and genetic data have implicated lipoprotein(a) as a potentially modifiable risk factor for atherosclerotic disease and aortic stenosis, but there are no approved pharmacological treatments. Objectives: To assess the safety, tolerability, pharmacokinetics, and effects of lepodisiran on lipoprotein(a) concentrations after single doses of the drug; lepodisiran is a short interfering RNA directed at hepatic synthesis of apolipoprotein(a), an essential component necessary for assembly of lipoprotein(a) particles. Design, Setting, and Participants: A single ascending-dose trial conducted at 5 clinical research sites in the US and Singapore that enrolled 48 adults without cardiovascular disease and with lipoprotein(a) serum concentrations of 75 nmol/L or greater (or ≥30 mg/dL) between November 18, 2020, and December 7, 2021; the last follow-up visit occurred on November 9, 2022. Interventions: Participants were randomized to receive placebo or a single dose of lepodisiran (4 mg, 12 mg, 32 mg, 96 mg, 304 mg, or 608 mg) administered subcutaneously. Main Outcomes and Measures: The primary outcome was the safety and tolerability of the single ascending doses of lepodisiran. The secondary outcomes included plasma levels of lepodisiran for 168 days after dose administration and changes in fasting lipoprotein(a) serum concentrations through a maximum follow-up of 336 days (48 weeks). Results: Of the 48 participants enrolled (mean age, 46.8 [SD, 11.6] years; 35% were women), 1 serious adverse event occurred. The plasma concentrations of lepodisiran reached peak levels within 10.5 hours and were undetectable by 48 hours. The median baseline lipoprotein(a) concentration was 111 nmol/L (IQR, 78 to 134 nmol/L) in the placebo group, 78 nmol/L (IQR, 50 to 152 nmol/L) in the 4 mg of lepodisiran group, 97 nmol/L (IQR, 86 to 107 nmol/L) in the 12-mg dose group, 120 nmol/L (IQR, 110 to 188 nmol/L) in the 32-mg dose group, 167 nmol/L (IQR, 124 to 189 nmol/L) in the 96-mg dose group, 96 nmol/L (IQR, 72 to 132 nmol/L) in the 304-mg dose group, and 130 nmol/L (IQR, 87 to 151 nmol/L) in the 608-mg dose group. The maximal median change in lipoprotein(a) concentration was -5% (IQR, -16% to 11%) in the placebo group, -41% (IQR, -47% to -20%) in the 4 mg of lepodisiran group, -59% (IQR, -66% to -53%) in the 12-mg dose group, -76% (IQR, -76% to -75%) in the 32-mg dose group, -90% (IQR, -94% to -85%) in the 96-mg dose group, -96% (IQR, -98% to -95%) in the 304-mg dose group, and -97% (IQR, -98% to -96%) in the 608-mg dose group. At day 337, the median change in lipoprotein(a) concentration was -94% (IQR, -94% to -85%) in the 608 mg of lepodisiran group. Conclusions and Relevance: In this phase 1 study of 48 participants with elevated lipoprotein(a) levels, lepodisiran was well tolerated and produced dose-dependent, long-duration reductions in serum lipoprotein(a) concentrations. The findings support further study of lepodisiran. Trial Registration: ClinicalTrials.gov Identifier: NCT04914546.

Small size apolipoprotein(a) isoforms enhance inflammatory and proteolytic potential of collagen-primed monocytes.

BACKGROUND: Atherosclerosis is an inflammatory process involving activation of monocytes recruited by various chemoattractant factors, among which lipoprotein(a) and its specific apolipoprotein apo(a). Lp(a) contains a specific apolipoprotein apo(a) which size is determined by a variable number of repeats of a specific structural domain, the kringle IV type 2 (IV-2). Lp(a) plasma concentration and apo(a) size is inversely correlated, and smaller apo(a) are major risk factors for coronary heart disease. DESIGN AND METHODS: The aim of this study was to evaluate the effect of recombinant apo(a) isoforms (containing 10, 18 or 34 kringles) on monocytes interacting with type I collagen. RESULTS: Apo(a) isoforms stimulated reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) production by monocytes, and not modified monocytes adhesion on type I collagen. This effect was specific of apo(a) since no effect was observed in the presence of plasminogen and was inversely related to apo(a) size. The lysine analogue 6-aminohexanoic acid which blocks the lysine binding sites (LBS), and carboxypeptidase B (CpB) which cleaves carboxy-terminal lysine residues, abolished apo(a)-induced ROS and MMP-9 production, highlighting an effect mediated by apo(a) lysing-binding sites. CONCLUSIONS: These results indicate that activation of collagen-primed monocytes stimulated with apo(a) is a Kringle number-dependent effect and reinforce the hypothesis of a role for small size apo(a) isoforms in atherothrombosis.

miR-23b-3p and miR-125b-5p downregulate apo(a) expression by targeting Ets1 in HepG2 cells.

High concentrations of plasma lipoprotein(a) [Lp(a)] have been inferred to be an independent risk factor for cardiovascular and cerebrovascular diseases, such as coronary artery diseases, restenosis, and stroke. Apolipoprotein(a) [apo(a)] is one of the most important components of Lp(a) and contributes greatly to the increased concentration of plasma Lp(a). As a critical positive transacting factor of apo(a) gene, Ets1 has been proven as a target gene of several miRNAs, such as miR-193b, miR-125b-5p, miR-200b, miR-1, and miR-499. In this study, a series of experiments on miRNAs and relative miRNAs inhibitor delivered HepG2 cells were conducted, and two miRNAs that downregulate the apo(a) by targeting the 3'-UTR of Ets1 were identified. Results showed that apo(a) and Ets1 were differentially expressed in SMMC7721 and HepG2 cell lines. Meanwhile, apo(a) and Ets1 were inversely correlated with several hepatic endogenous miRNAs, such as miR-125b-5p, miR-23b-3p, miR-26a-5p, and miR-423-5p, which were predicted to bind to Ets1. Results show that miR-125b-5p and miR-23b-3p mimics could inhibit the synthesis of apo(a) by directly targeting Ets1 in HepG2, thereby reducing the plasma Lp (a) concentration.

A long non-coding RNA, APOA4-AS, regulates APOA4 expression depending on HuR in mice.

Long non-coding RNAs (lncRNAs) have been shown to be critical biomarkers or therapeutic targets for human diseases. However, only a small number of lncRNAs were screened and characterized. Here, we identified 15 lncRNAs, which are associated with fatty liver disease. Among them, APOA4-AS is shown to be a concordant regulator of Apolipoprotein A-IV (APOA4) expression. APOA4-AS has a similar expression pattern with APOA4 gene. The expressions of APOA4-AS and APOA4 are both abnormally elevated in the liver of ob/ob mice and patients with fatty liver disease. Knockdown of APOA4-AS reduces APOA4 expression both in vitro and in vivo and leads to decreased levels of plasma triglyceride and total cholesterol in ob/ob mice. Mechanistically, APOA4-AS directly interacts with mRNA stabilizing protein HuR and stabilizes APOA4 mRNA. Deletion of HuR dramatically reduces both APOA4-AS and APOA4 transcripts. This study uncovers an anti-sense lncRNA (APOA4-AS), which is co-expressed with APOA4, and concordantly and specifically regulates APOA4 expression both in vitro and in vivo with the involvement of HuR.

H2 S inhibits apo(a) expression and secretion through PKCα/FXR and Akt/HNF4α pathways in HepG2 cells.

Lipoprotein(a) [Lp(a)] is a strong genetic risk factor for coronary heart diseases. However, the metabolism of this protein remains poorly understood. Efficient and specific drugs that can decrease high plasma levels of Lp(a) have not been developed yet. Hydrogen sulfide (H2 S), a member of the gas transmitter family, performs important biological actions, including protection against cardiovascular diseases and maintenance of the lipid metabolism equilibrium in hepatocytes and adipocytes. In this study, we investigated the possible molecular mechanism of H2 S that influences apolipoprotein(a) [apo(a)] biosynthesis. We also determined the effects of H2 S on apo(a) expression and secretion in HepG2 cells as well as the underlying mechanisms. Results showed that H2 S significantly inhibited the expression and secretion levels of apo(a). These effects were attenuated by the PKCα inhibitor and FXR siRNA. H2 S also reduced HNF4α expression and enhanced FXR expression. The Akt inhibitor partially reversed H2 S-induced inhibition of apo(a) and HNF4α expression and apo(a) secretion. This study reveals that H2 S suppressed apo(a) expression and secretion via the PKCα-FXR and PI3K/Akt-HNF4α pathways.

Differential expression of oxidation-specific epitopes and apolipoprotein(a) in progressing and ruptured human coronary and carotid atherosclerotic lesions.

The relationships between oxidation-specific epitopes (OSE) and lipoprotein (a) [Lp(a)] and progressive atherosclerosis and plaque rupture have not been determined. Coronary artery sections from sudden death victims and carotid endarterectomy specimens were immunostained for apoB-100, oxidized phospholipids (OxPL), apo(a), malondialdehyde-lysine (MDA), and MDA-related epitopes detected by antibody IK17 and macrophage markers. The presence of OxPL captured in carotid and saphenous vein graft distal protection devices was determined with LC-MS/MS. In coronary arteries, OSE and apo(a) were absent in normal coronary arteries and minimally present in early lesions. As lesions progressed, apoB and MDA epitopes did not increase, whereas macrophage, apo(a), OxPL, and IK17 epitopes increased proportionally, but they differed according to plaque type and plaque components. Apo(a) epitopes were present throughout early and late lesions, especially in macrophages and the necrotic core. IK17 and OxPL epitopes were strongest in late lesions in macrophage-rich areas, lipid pools, and the necrotic core, and they were most specifically associated with unstable and ruptured plaques. Specific OxPL were present in distal protection devices. Human atherosclerotic lesions manifest a differential expression of OSEs and apo(a) as they progress, rupture, and become clinically symptomatic. These findings provide a rationale for targeting OSE for biotheranostic applications in humans.

The role of apolipoprotein A5 in non-alcoholic fatty liver disease.

BACKGROUND: Apolipoprotein A5 (apoA5) is a recently described liver-specific protein that has been shown to influence triglyceride (TG) metabolism. ApoA5 transgenic mice display dramatically reduced TG levels, while in contrast apoA5 deficiency in humans was reported to result in marked hypertriglyceridemia. ApoA5 exerts its extracellular effects by increasing lipolysis of TG-rich lipoproteins, while in vitro data suggest additional intrahepatic effects. METHODS: In this study the authors set out to investigate a possible role of apoA5 in non-alcoholic fatty liver disease (NAFLD). We thus determined hepatic apoA5 expression in 15 obese subjects with histologically proven NAFLD undergoing bariatric surgery. In addition, the authors established a hepatic cell culture model of apoA5 knockdown by transfecting human hepatoma cells (HepG2) with apoA5 small interfering (si) RNA, and determined intracellular TG content and expression levels of key enzymes and transcription factors of intrahepatic lipid metabolism in these cells. RESULTS: Pronounced weight loss and associated histologically verified improvement of hepatic steatosis were accompanied by significant reductions of hepatic apoA5 mRNA expression levels. Significant apoA5 knockdown in HepG2 cells resulted in a marked decrease of intracellular TG content. When HepG2 cells were co-transfected with apoA5 and peroxisome proliferator-activated receptor gamma (PPARγ), reductions in hepatic TG accumulation were significantly less pronounced when compared to apoA5 siRNA transfected HepG2 cells. CONCLUSIONS: In obese subjects, hepatic apoA5 mRNA expression decreases after weight loss and improvements in hepatic steatosis. The authors' in vitro data demonstrate that apoA5 influences intrahepatic TG metabolism and that these intracellular effects of apoA5 are accompanied by changes in PPARγ mRNA expression. In summary, the data suggest that as well as several other factors, apoA5 might be involved in the pathogenesis of hepatic steatosis.

The apolipoprotein A-I gene is actively expressed in the rapidly myelinating avian peripheral nerve.

The expression of the apolipoprotein A-I (apo A-I) gene was investigated in the myelinating sciatic nerve. Hybridization analysis with an apo A-I cDNA probe obtained from a cDNA library of mRNA isolated from rapidly myelinating chick sciatic nerve indicated that apo A-I coding transcripts increase during development in the chick sciatic nerve in parallel with the increase of myelin lamellae. Substantial apo A-I-like immunoreactivity in chick sciatic nerve homogenates was detected by Western blotting. The amount of antigen increased from the 15-d embryonic stage to 1 d posthatch and then decreased. Two subcellular fractions corresponding to the cytoplasmic compartments were particularly enriched in apo A-I. apo A-I immunoreactivity was also found in highly purified myelin preparations. Immunohistochemical staining provided further evidence for the presence of apo A-I in the endoneurial compartment of the sciatic nerve. Electron microscopic examination of these fractions after negative staining showed the presence of spherical and disc-shaped particles resembling high density lipoproteins. The presence of apo A-I, cholesterol esters, phospholipids, and triacylglycerols in ultracentrifugal fractions corresponding to serum lipoproteins and the behavior of apo A-I on nondenaturing gradient gels implied that apo A-I was associated with lipid. Studies with short-term organ cultures of sciatic nerves from 1-d chicks strengthened the evidence for local synthesis and secretion of apo A-I and apo A-I-containing lipoproteins by this tissue. These results establish that the apo A-I gene is actively expressed in developing sciatic nerve during the period of rapid myelination. These findings support the hypothesis that apo A-I synthesized within the nerve participates in the local transport of lipids used in myelin biosynthesis.

Measurement of very low density and low density lipoprotein apolipoprotein (Apo) B-100 and high density lipoprotein Apo A-I production in human subjects using deuterated leucine. Effect of fasting and feeding.

Six normolipidemic male subjects, after an 8-h overnight fast, were given a bolus injection and then a 15-h constant intravenous infusion of [D3]L-leucine. Subjects were studied in the fasted state and on a second occasion in the fed state (small, physiological meals were given every hour for 15 h). Apolipoproteins were isolated by preparative gradient gel electrophoresis from plasma lipoproteins separated by sequential ultracentrifugation. Incorporation of [D3]L-leucine into apolipoproteins was monitored by negative ionization, gas chromatography-mass spectrometry. Production rates were determined by multiplying plasma apolipoprotein pool sizes by fractional production rates (calculated as the rate of isotopic enrichment [IE] of each protein as a fraction of IE achieved by VLDL (d less than 1.006 g/ml) apo B-100 at plateau. VLDL apo B-100 production was greater, and LDL (1.019 less than d less than 1.063 g/ml) apo B-100 production was less in the fed compared with the fasted state (9.9 +/- 1.7 vs. 6.4 +/- 1.7 mg/kg per d, P less than 0.01, and 8.9 +/- 1.2 vs. 13.1 +/- 1.2 mg/kg per d, P less than 0.05, respectively). No mean change was observed in high density lipoprotein apo A-I production. We conclude that: (a) this stable isotope, endogenous-labeling technique, for the first time allows for the in vivo measurement of apolipoprotein production in the fasted and fed state; and (b) since LDL apo B-100 production was greater than VLDL apo B-100 production in the fasted state, this study provides in vivo evidence that LDL apo B-100 can be produced independently of VLDL apo B-100 in normolipidemic subjects.

The role of lecithin: cholesterol acyltransferase for lipoprotein (a) assembly. Structural integrity of low density lipoproteins is a prerequisite for Lp(a) formation in human plasma.

The composition of lipoproteins in the plasma of patients with LCAT deficiency (LCAT-D) is grossly altered due to the lack of cholesteryl esters which form the core of normal lipoproteins. When plasma from LCAT-D patients and their relatives was examined we found that nine heterozygotes had plasma Lp(a) levels of 2-13 mg/dl whereas none of 11 affected homozygous individuals from different families contained detectable amounts of Lp(a) in their plasma. Therefore, the binding of apo(a) to LDL density particles was studied in vitro using LDL density fractions prepared from patients, and recombinant apo(a) [r-apo(a)], which was expressed and secreted by transfected COS-7 cells. The LDL from heterozygotes were chemically indistinguishable from normal LDL and homogeneous with regard to morphology, whereas the crude LDL floating fraction from homozygotes consisted of a heterogeneous mixture of large vesicles, and small spheres resembling normal LDL. The LDL density fraction from the LCAT-D patient lacked almost completely cholesteryl esters. Incubation of LCAT-D plasma with active LCAT caused a substantial augmentation of the original subfraction which morphologically resembled normal LDL. Using r-apo(a) and normal LDL or LDL of heterozygous individuals, apoB:r-apo(a) complexes were formed when incubated at 37 degrees C in vitro for 20 h. In contrast, the total LDL floating fraction from a homozygous LCAT-D patient failed to form apoB:r-apo(a) complexes. After treatment with active LCAT, a significant apoB:r-apo(a) association was observed with LCAT-D LDL-density particles. Our data emphasize the importance of the integrity of LDL structure and composition for the formation of Lp(a). In addition, we demonstrate that the absence of LCAT activity has a fundamental impact on the regulation of plasma Lp(a) levels.

Regulation of intestinal and hepatic apoprotein synthesis after chronic fat and cholesterol feeding.

Although diet influences levels of lipoproteins and their corresponding apoproteins, its effects on the molecular regulation of apoprotein synthesis are relatively unknown. Male Sprague-Dawley rats were fed an atherogenic diet containing cholesterol and propylthiouracil (PTU). Intestinal apo AI and AIV mRNA concentrations were decreased by the atherogenic diet, but apo AI and AIV synthesis was increased in vitro (organ explants) and in vivo (polysome runoff), consistent with regulation at the translational level. In contrast, hepatic apo E mRNA concentration and synthesis were increased after the atherogenic diet, consistent with pretranslational regulation. The response to cholesterol feeding for hepatic apo AI and E showed a third pattern of regulation, in which synthesis increased and mRNA content remained stable or fell, again suggesting translational control, but polysome runoff synthesis was unchanged. The apparent importance of translational regulation in the intestine is consistent with the necessity for the tissue to respond rapidly to changes in intraluminal content.

Altered regulation of apolipoprotein A-IV gene expression in the liver of the genetically obese Zucker rat.

Apolipoprotein (apo) A-IV, a structural component of chylomicrons and high-density lipoproteins, may play a role in the catabolism of triglyceride-rich lipoproteins and in reverse cholesterol transport. To study the regulation of apoA-IV gene expression by genetic and nutritional factors, we determined the effect of a fish oil-rich and a sucrose-rich diet on apoA-IV gene transcription and nuclear and total cellular apoA-IV mRNA abundance in livers of genetically obese, hyperlipoproteinemic (fa/fa) Zucker rats and their lean (Fa/-) littermates. In obese rats fed chow, hepatic apoA-IV gene expression was more than twofold higher than in lean rats because of a post-transcriptional mechanism. apoA-I gene expression and apoC-III mRNA levels, studied as controls, were similar in both groups. The fish oil-rich diet reduced total cellular apoA-IV mRNA abundance transcriptionally to 34 +/- 4% of basal values in lean rats, but did not alter apoA-IV gene expression in obese rats. In contrast, this diet reduced apoA-I gene expression in both lean and obese animals. The sucrose-rich diet increased apoA-IV gene expression twofold in both lean and obese rats. Thus, genetic obesity alters the response of hepatic apoA-IV gene expression to a lipid-lowering diet rich in fish oil by a mechanism affecting transcriptional regulation.

Mouse apolipoprotein A-IV gene: nucleotide sequence and induction by a high-lipid diet.

Apolipoprotein A-IV (apo A-IV) functions in conjunction with other apolipoproteins to form lipoprotein particles which are involved in lipid homeostasis. In this report we present the nucleotide sequence of the mouse apo A-IV gene and demonstrate its induction in the liver by chronically high dietary lipid. The apo A-IV gene consists of three exons and two introns. The introns separate evolutionarily conserved and functional polypeptide domains. Intron 1 divides most of the apo A-IV signal peptide from the amino terminus of the mature plasma protein. The second intron separates a highly evolutionarily conserved, variant amphipathic peptide repeat from the remainder of the mature apo A-IV protein. The 5' flanking region has several interesting features. The apo A-IV gene has variant TATA and CAT box sequences, TTTAAA and CCAACG, respectively. There are five G-rich direct repeats of 10 nucleotides and a short inverted repeat in the 5' flanking region. We speculate that these sequence elements in the 5' flanking region may be involved in the regulation of apo A-IV gene expression. We also show that chronically high dietary lipid induces liver apo A-IV levels 10-fold in C57BL/6 mice, a strain susceptible to atherosclerotic lesions, while we observed no induction in nonsusceptible BALB/c and C3H mice.

[Expression profiles of lipid metabolism-related genes in liver of apoE(-/-)/LDLR(-/-) mice].

OBJECTIVE: To explore the relationship between the expression characteristics of lipid metabolism-related genes in the liver and early atherosclerotic lesions in apolipoprotein E and low density lipoprotein receptor gene double knockout (apoE(-/-)/LDLR(-/-)) mice. METHODS: RT-PCR was used to detect the differential expression of lipid metabolism-related genes in the liver of apoE(-/-)/LDLR(-/-) and wild type (WT) mice. Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) level as well as aortic morphology were also analyzed. RESULTS: Among the 11 lipid metabolism-related genes, apolipoprotein B100 (apoB100) mRNA levels were significantly higher in apoE(-/-)/LDLR(-/-)mice compared with WT mice. At 14 days, 1, 2 and 3 months of age, the level of mRNA expression were 1.55, 1.47, 1.50 and 2.42 folds of those of the age matched WT mice respectively. The fatty acid transporter (FAT/CD36) mRNA expression levels were higher in 14-day and 3-month old mice at 1.30 and 1.35 folds of those of the age matched WT mice, respectively. Apolipoprotein A IV (apoA IV) and Apolipoprotein AV (apoAV) mRNA levels were significantly down-regulated (0.89 fold decrease in 14-day, and 0.90 folds decrease in 3-month, respectively). The mRNA expression levels of apolipoprotein AI (apo AI), apolipoprotein F (apo F), peroxidase proliferator-activated receptor alpha (PPAR-alpha), liver X receptor alpha (LXRalpha), angiopoietin-like protein 3 (ANGPTL3), acyl-coenzymeA oxidase 1 (ACOX1) and carnitine palmitoyl transferase 1 (CPT1) had no significant changes. Serum TC, TG and LDL-C were higher than those of age matched WT mice at 7, 2 and 30 folds, respectively. Furthermore, apoE(-/-)/LDLR(-/-) mice demonstrated typical early atherosclerotic lesions at sinus and root regions of aorta in an age dependent manner. CONCLUSION: Alterations of the expression of lipid metabolism-related genes in liver play important roles in the development of AS in the apoE(-/-)/LDLR(-/-) mice at early ages.

Distinct metabolism of apolipoproteins (a) and B-100 within plasma lipoprotein(a).

OBJECTIVES: Lipoprotein(a) [Lp(a)] is mainly similar in composition to LDL, but differs in having apolipoprotein (apo) (a) covalently linked to apoB-100. Our purpose was to examine the individual metabolism of apo(a) and apoB-100 within plasma Lp(a). MATERIALS AND METHODS: The kinetics of apo(a) and apoB-100 in plasma Lp(a) were assessed in four men with dyslipidemia [Lp(a) concentration: 8.9-124.7nmol/L]. All subjects received a primed constant infusion of [5,5,5-(2)H3] L-leucine while in the constantly fed state. Lp(a) was immunoprecipitated directly from whole plasma; apo(a) and apoB-100 were separated by gel electrophoresis; and isotopic enrichment was determined by gas chromatography/mass spectrometry. RESULTS: Multicompartmental modeling analysis indicated that the median fractional catabolic rates of apo(a) and apoB-100 within Lp(a) were significantly different at 0.104 and 0.263 pools/day, respectively (P=0.04). The median Lp(a) apo(a) production rate at 0.248nmol/kg·day(-1) was significantly lower than that of Lp(a) apoB-100 at 0.514nmol/kg·day(-1) (P=0.03). CONCLUSION: Our data indicate that apo(a) has a plasma residence time (11days) that is more than twice as long as that of apoB-100 (4days) within Lp(a), supporting the concept that apo(a) and apoB-100 within plasma Lp(a) are not catabolized from the bloodstream as a unit in humans in the fed state.

Expression and secretion of chicken apolipoprotein AI in transfected COS cells.

A full-length chicken apolipoprotein A-I (apoAI) cDNA has been cloned into an expression vector, pRSVapoAI. This plasmid was transfected into a monkey kidney (COS-1) cell line in order to study apolipoprotein-lipid assembly. Chicken apoAI is the major apolipoprotein of chicken high-density lipoprotein (HDL), which is less complex in apolipoprotein content than the HDL of human plasma. The transient transfected COS-1 cells synthesized and secreted authentic plasma apoAI. Under serum-free medium conditions, COS cells secreted only proapoAI. A small portion (15%) of the secreted apoAI floated at a density 1.07-1.20 g/ml. Upon incubation with fetal bovine serum at 10 degrees C, a majority of the apoAI was recovered in the HDL density (1.06-1.20 g/ml) region. Secreted apoAI was labeled when transfected COS cells were incubated with [U-14C]palmitate, but the incorporation of radioactivity was not the result of fatty acid acylation through ester bond formation. These results indicate that heterologous COS-1 cells are capable of synthesizing and secreting apoAI, and that intracellular association of apoAI with lipids is not necessary for secretion.

Human apolipoprotein A-IV gene expression is modulated by members of the nuclear hormone receptor superfamily.

Human apolipoprotein A-IV (apo A-IV) is essentially synthesized in intestine and to a lower extent in liver. The presence of detectable amounts of apo A-IV messenger in HepG2 and Caco-2 cells enables us to study the transcription regulation of the gene in these cells. A main hypersensitive site to DNase I is detected in the DNA of HepG2 nuclei, in a region situated about 3400 base pairs upstream from the cap site. Transient expression experiments in HepG2, Caco-2 and HeLa cells indicate that HNF-4 activates the transcription of a reporter gene through the -3500/+67 sequence of the apo A-IV gene. This activation is repressed by another transcription factor, Ear3/COUP-TF. On the contrary, HNF-1, whose expression is controlled by HNF-4, is not involved in the regulation of the reporter gene transcription through the -6700/+67 genome sequence. These results indicate that the apo A-IV gene expression is modulated by orphan ligand members of the superfamily of nuclear hormone receptors.

Regulation of apolipoprotein A-I synthesis in lymph-drained rats.

The effect of lymph diversion on plasma apolipoprotein A-I levels was studied. In lymph fistula rats apolipoprotein A-I levels in plasma stayed constant in spite of a loss of an equivalent of one-half plasma pool of apolipoprotein A-I per day through the lymph fistula. This indicates that synthesis of apolipoprotein A-I increases or that catabolism of apolipoprotein A-I decreases in a compensatory manner as intestinal apolipoprotein A-I is diverted. By using incorporation of [3H]leucine into newly synthesized apolipoprotein A-I it was shown that 2.6-times as much [3H]leucine was incorporated into apolipoprotein A-I in thoracic duct drained animals compared to controls. In experiments in which 125I-labeled HDL was injected intravenously into rats, it was shown that catabolism of HDL and apolipoprotein A-I was not decreased in lymph-drained rats. These data thus suggest that an increased synthesis of apolipoprotein A-I occurs when the intestinal contribution of apolipoprotein A-I diminishes. This is probably due to an increase in liver protein synthesis.

Intestinal synthesis and lymphatic secretion of apolipoprotein A-IV after cessation of duodenal fat infusion: mediation by bile.

We tested whether secretion of apolipoprotein (apo) A-IV depends upon intestinal triglyceride (TG) transport by comparing output kinetics of TG and apo A-IV during and after duodenal lipid infusion in lymph-fistula rats. Lipid infusion (triolein, 40 mumol/h, 8 h) produced increases in lymphatic TG and apo A-IV output. After 8 h, triolein infusate was replaced with glucose-saline; TG output returned to basal levels 4-5 h later. However, apo A-IV output continued at significantly elevated levels until 20 h after the start of the experiment. Bile diversion blocked this continued output of A-IV during the post-lipid period, and resulted in basal TG output that was 75% lower than in bile-intact rats. Return of bile or low-dose triolein infusion (5 mumol/h) into the intestine reversed these effects. There were no differences in hepatic synthesis or filtration of plasma A-IV into lymph between bile-intact and bile-diverted groups. Intestinal A-IV synthesis was elevated in both groups even during the post-lipid period. The results support the hypothesis that intestinal triglyceride transport drives apo A-IV secretion, and suggest the existence of a bile-dependent, post-translational mechanism for the control of lymphatic apo A-IV output.

Apo A-IV: an update on regulation and physiologic functions.

Apolipoprotein (apo) A-IV, first identified 28 years ago as a plasma lipoprotein moiety, is now known to participate in the regulation of various metabolic pathways. It is synthesized primarily in the enterocytes of the small intestine during fat absorption. After entry into the bloodstream, the 46-kDa glycoprotein apo A-IV appears associated with chylomicrons, high-density lipoproteins, and in the lipoprotein-free fraction. It has a role in lipid absorption, transport and metabolism, and may act as a post-prandial satiety signal, an anti-oxidant and a major factor in the prevention of atherosclerosis. After summarizing and discussing these functions for reader's comprehension, the current review focuses on the regulation of apo A-IV by nutrients, biliary components, drugs, hormones and gastrointestinal peptides. The understanding of the involved mechanisms that underline apo A-IV regulation may in the long run allow us to switch on its gene, which may confer multiple beneficial effects, including the protection from atherosclerosis.