Hepatic Apolipoprotein M (ApoM) Overexpression Stimulates Formation of Larger ApoM/Sphingosine 1-Phosphate-enriched Plasma High Density Lipoprotein

Apolipoprotein M (apoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a signaling molecule active in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulated larger nascent HDL formation, compared with cells that did not express apoM; however, the in vivo role of apoM in HDL metabolism remains poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated hepatocyte-specific apoM transgenic (APOM Tg) mice, which had an ∼3–5-fold increase in plasma apoM levels compared with wild-type mice. Although HDL cholesterol concentrations were similar to wild-type mice, APOM Tg mice had larger plasma HDLs enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase, and S1P. Despite the presence of larger plasma HDLs in APOM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to that in wild-type mice. APOM Tg mice had an ∼5-fold increase in plasma S1P, which was predominantly associated with larger plasma HDLs. Primary hepatocytes from APOM Tg mice generated larger nascent HDLs and displayed increased sphingolipid synthesis and S1P secretion. Inhibition of ceramide synthases in hepatocytes increased cellular S1P levels but not S1P secretion, suggesting that apoM is rate-limiting in the export of hepatocyte S1P. Our data indicate that hepatocyte-specific apoM overexpression generates larger nascent HDLs and larger plasma HDLs, which preferentially bind apoM and S1P, and stimulates S1P biosynthesis for secretion. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection and may have other as yet unidentified functions.     

Apolipoprotein M modulates erythrocyte efflux and tubular reabsorption of sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) mediates several cytoprotective functions of HDL. apoM acts as a S1P binding protein in HDL. Erythrocytes are the major source of S1P in plasma. After glomerular filtration, apoM is endocytosed in the proximal renal tubules. Human or murine HDL elicited time- and dose-dependent S1P efflux from erythrocytes. Compared with HDL of wild-type (wt) mice, S1P efflux was enhanced in the presence of HDL from apoM transgenic mice, but not diminished in the presence of HDL from apoM knockout (Apom^−/−) mice. Artificially reconstituted and apoM-free HDL also effectively induced S1P efflux from erythrocytes. S1P and apoM were not measurable in the urine of wt mice. Apom^−/− mice excreted significant amounts of S1P. apoM was detected in the urine of mice with defective tubular endocytosis because of knockout of the LDL receptor-related protein, chloride-proton exchanger ClC-5 (Clcn5^−/−), or the cysteine transporter cystinosin. Urinary levels of S1P were significantly elevated in Clcn5^−/− mice. In contrast to Apom^−/− mice, these mice showed normal plasma concentrations for apoM and S1P. In conclusion, HDL facilitates S1P efflux from erythrocytes by both apoM-dependent and apoM-independent mechanisms. Moreover, apoM facilitates tubular reabsorption of S1P from the urine, however, with no impact on S1P plasma concentrations.     

The signal peptide anchors apolipoprotein M in plasma lipoproteins and prevents rapid clearance of apolipoprotein M from plasma

Lipoproteins consist of lipids solubilized by apolipoproteins. The lipid-binding structural motifs of apolipoproteins include amphipathic alpha-helixes and beta-sheets. Plasma apolipoprotein (apo) M lacks an external amphipathic motif but, nevertheless, is exclusively associated with lipoproteins (mainly high density lipoprotein). Uniquely, however, apoM is secreted to plasma without cleavage of its hydrophobic NH(2)-terminal signal peptide. To test whether the signal peptide serves as a lipoprotein anchor for apoM in plasma, we generated mice expressing a mutated apoM(Q22A) cDNA in the liver (apoM(Q22A)-Tg mice (transgenic mice)) and compared them with mice expressing wild-type human apoM (apoM-Tg mice). The substitution of the amino acid glutamine 22 with alanine in apoM(Q22A) results in secretion of human apoM without a signal peptide. The human apoM mRNA level in liver and the amount of human apoM protein secretion from hepatocytes were similar in apoM-Tg and apoM(Q22A)-Tg mice. Nevertheless, human apoM was not detectable in plasma of apoM(Q22A)-Tg mice, whereas it was easily measured in the apoM-Tg mice. To examine the plasma metabolism, recombinant apoM lacking the signal peptide was produced in Escherichia coli and injected into wild-type mice. The apoM without signal peptide did not associate with lipoproteins and was rapidly cleared in the kidney. Accordingly, ligation of the kidney arteries in apoM(Q22A)-Tg mice resulted in rapid accumulation of human apoM in plasma. The data suggest that hydrophobic signal peptide sequences, if preserved upon secretion, can anchor plasma proteins in lipoproteins. In the case of apoM, this mechanism prevents rapid loss by filtration in the kidney.     

Apolipoprotein M expression increases the size of nascent pre�� HDL formed by ATP binding cassette transporter A1

Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for preβ HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent preβ HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that ∼50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent preβ HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with ¹²⁵I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Preβ HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent preβ HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with preβ HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent preβ HDL formation but does stimulate the formation of larger-sized preβ HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto preβ HDL during or after their formation by ABCA1.     

Induction of insulin secretion by apolipoprotein M,a carrier for sphingosine 1-phosphate

Backgrounds

High-density lipoprotein (HDL) has been proposed to enhance β-cell functions. Clinical studies have suggested that apolipoprotein M (apoM), which rides mainly on HDL, is involved in diabetes; however, the underlying mechanism has not yet been elucidated. Recently, apoM was shown to be a carrier for sphingosine 1-phosphate (S1P), a bioactive lipid mediator. In the present study, we investigated the modulation of insulin secretion by apoM through the action of S1P.

Methods and results

We overexpressed apoM in the livers of C57BL6 mice using adenovirus gene transfer and found that the blood glucose levels under ad libitum feeding conditions were lower in the apoM-overexpressing mice. While an insulin tolerance test revealed that insulin sensitivity was not significantly affected, a glucose tolerance test revealed that apoM-overexpressing mice had a better glucose tolerance because of enhanced insulin secretion, a phenomenon that was reversed by treatment with VPC 23019, an antagonist against S1P1 and S1P3 receptor. In vitro experiments with MIN6 cells also revealed that apoM-containing lipoproteins enhanced insulin secretion, which was again inhibited by VPC 23019. ApoM retarded the degradation of S1P, and an increase in Pdx1 expression, the attenuation of endoreticulum stress, and the phosphorylation of Akt, AmpK, and Erk were observed as possible underlying mechanisms for the effect of S1P, maintained at a high concentration by apoM, on the increase in insulin secretion.

Conclusions

ApoM augmented insulin secretion by maintaining the S1P concentration under both in vivo and in vitro conditions.     

Apolipoprotein M associates to lipoproteins through its retained signal peptide

Apolipoprotein M (apoM) is predominantly associated with HDL. In this study, it was investigated whether apoM's uncleaved signal peptide is necessary for the protein's ability to associate with lipoproteins. ApoM with a cleavable signal peptide, Q22A, was expressed, together with wild-type apoM, in HEK293 cells. On size-exclusion chromatography, the elution profile of wild-type apoM was similar to that of human HDL-associated plasma apoM. In contrast, the size of the Q22A mutant corresponded to free, unassociated apoM. This strongly indicates that the signal peptide is indeed necessary for apoM's ability to associate with lipid.     

Comprehensive lipidomics in apoM-/- mice reveals an overall state of metabolic distress and attenuated hepatic lipid secretion into the circulation

Apolipoprotein M (apoM) participates in both high-density lipoprotein and cholesterol metabolism. Little is known about how apoM affects lipid composition of the liver and serum. In this study, we systemically investigated the effects of apoM on liver and plasma lipidomes and how apoM participates in lipid cycling, via apoM knockout in mice and the human SMMC-7721 cell line. We used integrated mass spectrometry-based lipidomics approaches to semiquantify more than 600 lipid species from various lipid classes, which include free fatty acids, glycerolipids, phospholipids, sphingolipids, glycosphingolipids, cholesterol, and cholesteryl esters (CEs), in apoM^-/- mouse. Hepatic accumulation of neutral lipids, including CEs, triacylglycerols, and diacylglycerols, was observed in apoM^-/- mice; while serum lipidomic analyses showed that, in contrast to the liver, the overall levels of CEs and saturated/monounsaturated fatty acids were markedly diminished. Furthermore, the level of ApoB-100 was dramatically increased in the liver, whereas significant reductions in both ApoB-100 and low-density lipoprotein (LDL) cholesterol were observed in the serum of apoM^-/- mice, which indicated attenuated hepatic LDL secretion into the circulation. Lipid profiles and proinflammatory cytokine levels indicated that apoM^-/- leads to hepatic steatosis and an overall state of metabolic distress. Taken together, these results revealed that apoM knockout leads to hepatic steatosis, impaired lipid secretion, and an overall state of metabolic distress.     

A novel role for ABCA1-generated large pre-�� migrating nascent HDL in the regulation of hepatic VLDL triglyceride secretion

In Tangier disease, absence of ATP binding cassette transporter A1 (ABCA1) results in reduced plasma HDL and elevated triglyceride (TG) levels. We hypothesized that hepatocyte ABCA1 regulates VLDL TG secretion through nascent HDL production. Silencing of ABCA1 expression in oleate-stimulated rat hepatoma cells resulted in: 1) decreased large nascent HDL (>10 nm diameter) and increased small nascent HDL (<10 nm) formation, 2) increased large buoyant VLDL1 particle secretion, and 3) decreased phosphatidylinositol-3 (PI3) kinase activation. Nascent HDL-containing conditioned medium from rat hepatoma cells or HEK293 cells transfected with ABCA1 was effective in increasing PI3 kinase activation and reducing VLDL TG secretion in ABCA1-silenced hepatoma cells. Addition of isolated large nascent HDL particles to ABCA1-silenced hepatoma cells inhibited VLDL TG secretion to a greater extent than small nascent HDL. Similarly, addition of recombinant HDL, but not human plasma HDL, was effective in attenuating TG secretion and increasing PI3 kinase activation in ABCA1-silenced cells. Collectively, these data suggest that large nascent HDL particles, assembled by hepatic ABCA1, generate a PI3 kinase-mediated autocrine signal that attenuates VLDL maturation and TG secretion. This pathway may explain the elevated plasma TG concentration that occurs in most Tangier subjects and may also account, in part, for the inverse relationship between plasma HDL and TG concentrations in individuals with compromised ABCA1 function.     

Apolipoprotein M promotes mobilization of cellular cholesterol in vivo

Objective

The HDL associated apolipoprotein M (apoM) protects against experimental atherosclerosis but the mechanism is unknown. ApoM increases preβ-HDL formation. We explored whether plasma apoM affects mobilization of cholesterol from peripheral cells in mice.

Methods and results

ApoM-enriched HDL from apoM-transgenic mice increased the in vitro efflux of ³H-cholesterol from macrophages by 24 ± 3% (p < 0.05) as compared with HDL from wild type (WT) mice, thus confirming previous findings. However, apoM-free HDL was not poorer than that of WT HDL to mobilize ³H-cholesterol. ³H-cholesterol-labeled foam cells were implanted in the peritoneal cavity of apoM^−/−, WT and apoM-transgenic mice to assess the mobilization of cholesterol from foam cells in vivo and subsequent excretion into feces. The results showed a statistically non-significant trend towards increased mobilization of cellular cholesterol to plasma with increasing plasma apoM. However, the apoM-genotype did not affect the excretion of ³H-cholesterol in feces. Nevertheless, when apoM^−/−, apoM-transgenic and WT mice received a constant intravenous infusion of ¹³C₂-cholesterol/intralipid for 5 h, the rate of enrichment of blood free cholesterol with free ¹³C₂-cholesterol was significantly lower (consistent with an increase in flux of unlabeled free cholesterol into the plasma) in the apoM-transgenic (3.0 ± 0.9‰/h) as compared to WT (5.7 ± 0.9‰/h, p < 0.05) and apoM^−/− (6.5 ± 0.6‰/h, p < 0.01) mice.

Conclusion

The present data indicate that the plasma apoM levels modulate the ability of plasma to mobilize cellular cholesterol, whereas apoM has no major effect on the excretion of cholesterol into feces.     

Sphingosine 1‐phosphate and its carrier apolipoprotein M in human sepsis and in Escherichia coli sepsis in baboons

Sphingosine 1‐phosphate (S1P) is an important regulator of vascular integrity and immune cell migration, carried in plasma by high‐density lipoprotein (HDL)‐associated apolipoprotein M (apoM) and by albumin. In sepsis, the protein and lipid composition of HDL changes dramatically. The aim of this study was to evaluate changes in S1P and its carrier protein apoM during sepsis. For this purpose, plasma samples from both human sepsis patients and from an experimental Escherichia coli sepsis model in baboons were used. In the human sepsis cohort, previously studied for apoM, plasma demonstrated disease‐severity correlated decreased S1P levels, the profile mimicking that of plasma apoM. In the baboons, a similar disease‐severity dependent decrease in plasma levels of S1P and apoM was observed. In the lethal E. coli baboon sepsis, S1P decreased already within 6–8 hrs, whereas the apoM decrease was seen later at 12–24 hrs. Gel filtration chromatography of plasma from severe human or baboon sepsis on Superose 6 demonstrated an almost complete loss of S1P and apoM in the HDL fractions. S1P plasma concentrations correlated with the platelet count but not with erythrocytes or white blood cells. The liver mRNA levels of apoM and apoA1 decreased strongly upon sepsis induction and after 12 hr both were almost completely lost. In conclusion, during septic challenge, the plasma levels of S1P drop to very low levels. Moreover, the liver synthesis of apoM decreases severely and the plasma levels of apoM are reduced. Possibly, the decrease in S1P contributes to the decreased endothelial barrier function observed in sepsis.     

Nascent HDL formation in hepatocytes and role of ABCA1, ABCG1, and SR-BI

To study the mechanisms of hepatic HDL formation, we investigated the roles of ABCA1, ABCG1, and SR-BI in nascent HDL formation in primary hepatocytes isolated from mice deficient in ABCA1, ABCG1, or SR-BI and from wild-type (WT) mice. Under basal conditions, in WT hepatocytes, cholesterol efflux to exogenous apoA-I was accompanied by conversion of apoA-I to HDL-sized particles. LXR activation by T0901317 markedly enhanced the formation of larger HDL-sized particles as well as cellular cholesterol efflux to apoA-I. Glyburide treatment completely abolished the formation of 7.4 nm diameter and greater particles but led to the formation of novel 7.2 nm-sized particles. However, cells lacking ABCA1 failed to form such particles. ABCG1-deficient cells showed similar capacity to efflux cholesterol to apoA-I and to form nascent HDL particles compared with WT cells. Cholesterol efflux to apoA-I and nascent HDL formation were slightly but significantly enhanced in SR-BI-deficient cells compared with WT cells under basal but not LXR activated conditions. As in WT but not in ABCA1-deficient hepatocytes, 7.2 nm-sized particles generated by glyburide treatment were also detected in ABCG1-deficient and SR-BI-deficient hepatocytes. Our data indicate that hepatic nascent HDL formation is highly dependent on ABCA1 but not on ABCG1 or SR-BI.     

LDL Receptor and ApoE Are Involved in the Clearance of ApoM-associated Sphingosine 1-Phosphate

Sphingosine 1-phosphate (S1P) is a vasoactive lipid mediator that is speculated to be involved in various aspects of atherosclerosis. About 70% of circulating plasma S1P is carried on HDL, and several pleiotropic properties of HDL have been ascribed to S1P. In the previous study with human subjects, however, LDL cholesterol or apoB, but not HDL cholesterol or apoA-I, had a significant positive correlation with the plasma S1P level, suggesting that the metabolic pathway for LDL might have some roles in the metabolism of S1P. In this study, we analyzed the association between LDL receptor, an important protein in the clearance of LDL, and circulating S1P. We observed that in LDL receptor-overexpressing mice, the plasma S1P levels as well as apolipoprotein M (apoM), a carrier of S1P, were decreased and that exogenously administered C₁₇S1P bound to apoM-containing lipoproteins was cleared more rapidly. Unlike the situation in wild-type mice, LDL receptor overexpression in apoE-deficient mice did not reduce the plasma S1P or apoM levels, suggesting that apoE might be a ligand for the LDL receptor during the clearance of these factors. The present findings clarify the novel roles of the LDL receptor and apoE in the clearance of S1P, a multifunctional bioactive phospholipid.     

Critical role of ABCA1 transporter in sphingosine 1-phosphate release from astrocytes

Sphingosine 1-phosphate (S1P) is accumulated in lipoproteins, especially high-density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S1P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL-like lipoprotein formation, stimulated extracellular S1P accumulation in the presence of its precursor sphingosine. The released S1P was present together with apoE particles in the HDL fraction. S1P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATP-binding cassette transporter A1 (ABCA1). Astrocytes from Abca1−/− mice also showed impairment of retinoic acid/dibutyryl cAMP-induced S1P release in association with the blockage of HDL-like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up-regulation of ABCA1 was requisite to stimulate S1P release. We conclude that the S1P release from astrocytes is coupled with lipoprotein formation through ABCA1.     

Implications of cerebrovascular ATP-binding cassette transporter G1 (ABCG1) and apolipoprotein M in cholesterol transport at the blood-brain barrier

Impaired cholesterol/lipoprotein metabolism is linked to neurodegenerative diseases such as Alzheimer's disease (AD). Cerebral cholesterol homeostasis is maintained by the highly efficient blood-brain barrier (BBB) and flux of the oxysterols 24(S)-hydroxycholesterol and 27-hydroxycholesterol, potent liver-X-receptor (LXR) activators. HDL and their apolipoproteins are crucial for cerebral lipid transfer, and loss of ATP binding cassette transporters (ABC)G1 and G4 results in toxic accumulation of oxysterols in the brain. The HDL-associated apolipoprotein (apo)M is positively correlated with pre-β HDL formation in plasma; its presence and function in the brain was thus far unknown. Using an in vitro model of the BBB, we examined expression, regulation, and functions of ABCG1, ABCG4, and apoM in primary porcine brain capillary endothelial cells (pBCEC). RT Q-PCR analyses and immunoblotting revealed that in addition to ABCA1 and scavenger receptor, class B, type I (SR-BI), pBCEC express high levels of ABCG1, which was up-regulated by LXR activation. Immunofluorescent staining, site-specific biotinylation and immunoprecipitation revealed that ABCG1 is localized both to early and late endosomes and on apical and basolateral plasma membranes. Using siRNA interference to silence ABCG1 (by 50%) reduced HDL-mediated [³H]-cholesterol efflux (by 50%) but did not reduce [³H]-24(S)-hydroxycholesterol efflux. In addition to apoA-I, pBCEC express and secrete apoM mainly to the basolateral (brain) compartment. HDL enhanced expression and secretion of apoM by pBCEC, apoM-enriched HDL promoted cellular cholesterol efflux more efficiently than apoM-free HDL, while apoM-silencing diminished cellular cholesterol release. We suggest that ABCG1 and apoM are centrally involved in regulation of cholesterol metabolism/turnover at the BBB.     

Increased plasma apoM levels impair triglyceride turnover in mice

ObjectiveApolipoprotein M (apoM) is an essential transporter of plasma Sphingosine-1-Phosphate (S1P), typically attached to all lipoprotein classes, but with a majority bound to high density lipoproteins (HDL). ApoM-deficient mice display an increased activity in brown adipose tissue and a concomitant fast turnover of triglycerides. In what manner apoM/S1P affect the triglyceride metabolism is however still unknown and explored in the present study.MethodsTriglyceride turnover and potentially associated metabolic pathways were studied in the female human apoM transgenic mouse model (apoM-Tg) with increased plasma apoM and S1P levels. The model was compared with wild type (WT) mice.ResultsApoM-Tg mice had a reduced plasma triglyceride turnover rate and a lower free fatty acid uptake in subcutaneous adipocytes compared to WT mice. Screening for potential molecular mechanisms furthermore revealed a reduction in plasma lipase activity in apoM-Tg animals. Overexpression of apoM also reduced the plasma levels of fibroblast growth factor 21 (FGF21).ConclusionsThe study features the significant role of the apoM/S1P axis in maintaining a balanced triglyceride metabolism. Further, it also highlights the risk of inducing dyslipidaemia in patients receiving S1P-analouges and additionlly emphasizes the apoM/S1P axis as a potential therapeutic target in treatment of hypertriglyceridemia.     

Isolation and characterization of human apolipoprotein M-containing lipoproteins

Apolipoprotein M (apoM) is a novel apolipoprotein with unknown function. In this study, we established a method for isolating apoM-containing lipoproteins and studied their composition and the effect of apoM on HDL function. ApoM-containing lipoproteins were isolated from human plasma with immunoaffinity chromatography and compared with lipoproteins lacking apoM. The apoM-containing lipoproteins were predominantly of HDL size; approximately 5% of the total HDL population contained apoM. Mass spectrometry showed that the apoM-containing lipoproteins also contained apoJ, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III, paraoxonase 1, and apoB. ApoM-containing HDL (HDL(apoM+)) contained significantly more free cholesterol than HDL lacking apoM (HDL(apoM-)) (5.9 +/- 0.7% vs. 3.2 +/- 0.5%; P < 0.005) and was heterogeneous in size with both small and large particles. HDL(apoM+) inhibited Cu(2+)-induced oxidation of LDL and stimulated cholesterol efflux from THP-1 foam cells more efficiently than HDL(apoM-). In conclusion, our results suggest that apoM is associated with a small heterogeneous subpopulation of HDL particles. Nevertheless, apoM designates a subpopulation of HDL that protects LDL against oxidation and stimulates cholesterol efflux more efficiently than HDL lacking apoM.     

Serendipitous Fatty Acid Binding Reveals the Structural Determinants for Ligand Recognition in Apolipoprotein M

Apolipoprotein M (ApoM) is a 25-kDa HDL-associated apolipoprotein and a member of the lipocalin family of proteins. Mature apoM retains its signal peptide, which serves as a lipid anchor attaching apoM to the lipoproteins, thereby keeping it in the circulation. Studies in mice have suggested apoM to be antiatherogenic, but its physiological function is yet unknown. We have now determined the 1.95 Å resolution crystal structure of recombinant human apoM expressed in Escherichia coli and made the unexpected discovery that apoM, although refolded from inclusion bodies, was in complex with fatty acids containing 14, 16 or 18 carbon atoms. ApoM displays the typical lipocalin fold characterised by an eight-stranded antiparallel β-barrel that encloses an internal ligand-binding pocket. The crystal structures of two different complexes provide a detailed picture of the ligand-binding determinants of apoM. Additional fatty acid- and lipid-binding studies with apoM and the mutants apoM^W47F and apoM^W100F showed that sphingosine-1-phosphate is able to displace the bound fatty acids and efficiently quenched the intrinsic fluorescence with an IC₅₀ of 0.90 μM. Whereas the fatty acids bound in the crystal structure could be a mere consequence of recombinant protein production, the observed binding of sphingosine-1-phosphate might provide a key to a better understanding of the physiological function of apoM.     

The plasma concentration of HDL-associated apoM is influenced by LDL receptor-mediated clearance of apoB-containing particles

ApoM is mainly associated with HDL. Nevertheless, we have consistently observed positive correlations of apoM with plasma LDL cholesterol in humans. Moreover, LDL receptor deficiency is associated with increased plasma apoM in mice. Here, we tested the idea that plasma apoM concentrations are affected by the rate of LDL receptor-mediated clearance of apoB-containing particles. We measured apoM in humans each carrying one of three different LDL receptor mutations (n = 9) or the apoB3500 mutation (n = 12). These carriers had increased plasma apoM (1.34 ± 0.13 μM, P = 0.003, and 1.23 ± 0.10 μM, P = 0.02, respectively) as compared with noncarriers (0.93 ± 0.04 μM). When we injected human apoM-containing HDL into Wt (n = 6) or LDL receptor-deficient mice (n = 6), the removal of HDL-associated human apoM was delayed in the LDL receptor-deficient mice. After 2 h, 54 ± 5% versus 90 ± 8% (P < 0.005) of the initial amounts of human apoM remained in the plasma of Wt and LDL receptor-deficient mice, respectively. Finally, we compared the turnover of radio-iodinated LDL and plasma apoM concentrations in 45 normocholesterolemic humans. There was a negative correlation between plasma apoM and the fractional catabolic rate of LDL (r = -0.38, P = 0.009). These data suggest that the plasma clearance of apoM, despite apoM primarily being associated with HDL, is influenced by LDL receptor-mediated clearance of apoB-containing particles.     

Protein unfolding allows use of commercial antibodies in an apolipoprotein M sandwich ELISA

apoM is a member of the lipocalin superfamily and circulates in plasma attached to HDL particles. apoM plays a role in cholesterol metabolism and has recently been identified as transporter for the signaling lipid, sphingosine-1-phosphate (S1P), in plasma. S1P is implicated in several inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. The ability to accurately measure apoM is crucial for investigating its biological functions and possible clinical implications. However, reliable commercial methods have been lacking so far. Therefore, we have developed an assay that specifically recognizes human apoM in plasma using commercially available reagents. Commercial apoM antibodies were screened for compatibility in a sandwich ELISA-based assay. One optimal pair of antibodies was chosen, and sample preparation, buffers, and incubation times were optimized to generate a simple and reproducible method. Validation and comparison to a previously described ELISA for apoM confirmed that the assay displays a high degree of sensitivity, specificity, and precision. Our results show that commercially available antibodies can be used to accurately measure human plasma apoM. This method can be implemented in every laboratory and will help promote high quality research.