apoM Concentrations Research Articles

Sphingosine 1-Phosphate and Apolipoprotein M Levels and Their Correlations with Inflammatory Biomarkers in Patients with Untreated Familial Hypercholesterolemia.

High-density lipoprotein (HDL)-bound apolipoprotein M/sphingosine 1-phosphate (ApoM/S1P) complex in cardiovascular diseases serves as a bridge between HDL and endothelial cells, maintaining a healthy endothelial barrier. To date, S1P and ApoM in patients with untreated heterozygous familial hypercholesterolemia (HeFH) have not been extensively studied. Eighty-one untreated patients with HeFH and 32 healthy control subjects were included in this study. Serum S1P, ApoM, sCD40L, sICAM-1, sVCAM-1, oxLDL, and TNFα concentrations were determined by ELISA. PON1 activities were measured spectrophotometrically. Lipoprotein subfractions were detected by Lipoprint. We diagnosed FH using the Dutch Lipid Clinic Network criteria. Significantly higher serum S1P and ApoM levels were found in HeFH patients compared to controls. S1P negatively correlated with large HDL and positively with small HDL subfractions in HeFH patients and the whole study population. S1P showed significant positive correlations with sCD40L and MMP-9 levels and PON1 arylesterase activity, while we found significant negative correlation between sVCAM-1 and S1P in HeFH patients. A backward stepwise multiple regression analysis showed that the best predictors of serum S1P were large HDL subfraction and arylesterase activity. Higher S1P and ApoM levels and their correlations with HDL subfractions and inflammatory markers in HeFH patients implied their possible role in endothelial protection.

Sphingosine-1-phosphate: metabolism, transport, atheroprotection and effect of statin treatment.

To better define the metabolism of sphingosine-1-phosphate (S1P), its transport in plasma and its interactions with S1P receptors on vascular cells, and to evaluate the effect of statin treatment on the subnormal plasma levels of high-density lipoprotein (HDL)-bound S1P characteristic of the atherogenic dyslipidemia of metabolic syndrome (MetS). Neither clinical intervention trials targeted to raising high-density lipoprotein-cholesterol (HDL-C) levels nor human genome-wide association studies (GWAS) studies have provided evidence to support an atheroprotective role of HDL. Recently however a large monogenic univariable Mendelian randomization on the N396S mutation in the gene encoding endothelial lipase revealed a causal protective effect of elevated HDL-C on coronary artery disease conferred by reduced enzyme activity. Given the complexity of the HDL lipidome and proteome, components of HDL other than cholesterol may in all likelihood contribute to such a protective effect. Among HDL lipids, S1P is a bioactive sphingolipid present in a small proportion of HDL particles (about 5%); indeed, S1P is preferentially enriched in small dense HDL3. As S1P is bound to apolipoprotein (apo) M in HDL, such enrichment is consistent with the elevated apoM concentration in HDL3. When HDL/apoM-bound S1P acts on S1P1 or S1P3 receptors in endothelial cells, potent antiatherogenic and vasculoprotective effects are exerted; those exerted by albumin-bound S1P at these receptors are typically weaker. When HDL/apoM-bound S1P binds to S1P2 receptors, proatherogenic effects may potentially be induced. Subnormal plasma levels of HDL-associated S1P are typical of dyslipidemic individuals at high cardiovascular risk and in patients with coronary heart disease. International Guidelines recommend statin treatment as first-line lipid lowering therapy in these groups. The cardiovascular benefits of statin therapy are derived primarily from reduction in low-density lipoprotein (LDL)-cholesterol, although minor contributions from pleiotropic actions cannot be excluded. Might statin treatment therefore normalize, directly or indirectly, the subnormal levels of S1P in dyslipidemic subjects at high cardiovascular risk? Our unpublished findings in the CAPITAIN study (ClinicalTrials.gov: NCT01595828), involving a cohort of obese, hypertriglyceridemic subjects (n = 12) exhibiting the MetS, showed that pitavastatin calcium (4 mg/day) treatment for 180days was without effect on either total plasma or HDL-associated S1P levels, suggesting that statin-mediated improvement of endothelial function is not due to normalization of HDL-bound S1P. Statins may however induce the expression of S1P1 receptors in endothelial cells, thereby potentiating increase in endothelial nitric oxide synthase response to HDL-bound S1P, with beneficial downstream vasculoprotective effects. Current evidence indicates that S1P in small dense HDL3 containing apoM exerts antiatherogenic effects and that statins exert vasculoprotective effects through activation of endothelial cell S1P1 receptors in response to HDL/apoM-bound S1P.

Apolipoprotein M and Risk of Type 2 Diabetes

Recent studies have discovered a role of apolipoprotein M (apoM) in energy metabolism, and observational analyses in humans suggest an association with type 2 diabetes. The causal relationship remains however elusive. To investigate whether reduced plasma apoM concentrations are causally linked to increased risk of type 2 diabetes. Prospective study design analyzed by Mendelian randomization. Two cohorts reflecting the Danish general population: the Copenhagen City Heart Study (CCHS, n = 8589) and the Copenhagen General Population Study (CGPS; n = 93 857). Observational analyses included a subset of participants from the CCHS with available plasma apoM (n = 725). Genetic analyses included the complete cohorts (n = 102 446). During a median follow-up of 16 years (CCHS) and 8 years (CGPS), 563 and 2132 participants developed type 2 diabetes. Plasma apoM concentration, genetic variants in APOM, and type 2 diabetes. First, we identified an inverse correlation between plasma apoM and risk of type 2 diabetes in a subset of participants from the CCHS (hazard ratio between highest vs lowest quartile (reference) = 0.32; 95% confidence interval = 0.1-1.01; P for trend = .02). Second, genotyping of specific single nucleotide polymorphisms in APOM further revealed a 10.8% (P = 6.2 × 10-5) reduced plasma apoM concentration in participants with variant rs1266078. Third, a meta-analysis including data from 599 451 individuals showed no association between rs1266078 and risk of type 2 diabetes. The present study does not appear to support a causal association between plasma apoM and risk of type 2 diabetes.

Apolipoprotein M Serum Levels Correlate with IgA Vasculitis and IgA Vasculitis Nephritis.

Objective IgA vasculitis (lgAV) is the most frequent vessel vasculitis in children, and the prognosis is related to the children's age and degree of nephritis. This study is aimed at investigating serum apolipoprotein M (apoM) levels in patients with lgAV patients and at evaluating the association between apoM and disease severity. Methods A total of 109 lgAV patients and 76 age- and sex-matched healthy controls were included. The age and gender of the study participants were matched. ApoM levels were measured by an enzyme-linked immunosorbent assay. Additionally, the serum levels of lipids, apolipoproteins, kidney biochemical profiles, immunoglobulins (IgA, IgG, IgM, and IgE), and the complements (C3 and C4) were assessed using an automatic biochemical analyzer. Results ApoM was increased significantly in lgAV patients compared to healthy controls. ApoM, meanwhile, was lower in patients with nephritis than in those without nephritis. The apoM levels were higher in classes I and II IgA vasculitis nephritis (lgAVN) patients than in classes III and IV. Besides, the apoM serum level < 24.81 mg/L was an independent predictive factor for lgAVN and can be independently associated with the presence of nephritis in lgAV patients. Meanwhile, the serum apoM concentration negatively correlated with the ISKDC grading score in lgAVN patients. Conclusions Serum apoM was elevated in lgAV patients and decreased gradually with the ISKDC grading score. ApoM (OR = 0.32, 95%CI = 0.12‐0.85, p = 0.023) was identified as a protective factor for nephritis in all lgAV patients.

Plasma apoM and S1P levels are inversely associated with mortality in African Americans with type 2 diabetes mellitus

apoM is a minor HDL apolipoprotein and carrier for sphingosine-1-phosphate (S1P). HDL apoM and S1P concentrations are inversely associated with atherosclerosis progression in rodents. We evaluated associations between plasma concentrations of S1P, plasma concentrations of apoM, and HDL apoM levels with prevalent subclinical atherosclerosis and mortality in the African American-Diabetes Heart Study participants (N = 545). Associations between plasma S1P, plasma apoM, and HDL apoM with subclinical atherosclerosis and mortality were assessed using multivariate parametric, nonparametric, and Cox proportional hazards models. At baseline, participants' median (25th percentile, 75th percentile) age was 55 (49, 62) years old and their coronary artery calcium (CAC) mass score was 26.5 (0.0, 346.5). Plasma S1P, plasma apoM, and HDL apoM were not associated with CAC. After 64 (57.6, 70.3) months of follow-up, 81 deaths were recorded. Higher concentrations of plasma S1P [odds ratio (OR) = 0.14, P = 0.01] and plasma apoM (OR = 0.10, P = 0.02), but not HDL apoM (P = 0.89), were associated with lower mortality after adjusting for age, sex, statin use, CAC, kidney function, and albuminuria. We conclude that plasma S1P and apoM concentrations are inversely and independently associated with mortality, but not CAC, in African Americans with type 2 diabetes after accounting for conventional risk factors.

Preeclampsia is associated with changes in the composition and dysfunction of high-density lipoproteins

Preeclampsia (PE) is a hypertensive disorder of pregnancy associated with abnormal placentation leading to poor placental perfusion, oxidative stress, and inflammation, which then leads to the dysfunction of the maternal endothelium. High density lipoproteins (HDL) exert protective effects on the vascular endothelium, especially by exerting an antioxidant effect and by stimulating the NO (nitric oxide) production by a mechanism involving sphingosine-1 phosphate (S1P). However, HDL can become dysfunctional and lose these protective properties. An increase in low-density lipoprotein (LDL) oxidation and a decrease in NO production have been described in PE. We hypothesized that HDL become dysfunctional in PE and fail to protect the maternal endothelium with, in particular, a loss of their antioxidant properties, explaining the increase of LDL oxidation and an S1P depletion explaining the decrease in NO production. We conducted a case-control study in 10 pregnant women with early severe PE matched by age and gestational age to 10 control healthy pregnant women. We isolated HDL from the serum of patients and control women by ultracentrifugation. In order to evaluate the antioxidant effect of HDL, we incubated human endothelial cells with the purified HDL, LDL and CuSO 4 for 6 hours. Then, we measured TBARs (ThioBarbituric Acid Reactive Substances) produced in cell culture supernatants. We have also measured the concentrations of S1P and apoM (apolipoprotein M), the apolipoprotein which binds S1P in HDL, by LC-MS-MS. The TBARs levels in culture supernatants were significantly raised when the endothelial cells were incubated with HDL purified from PE women rather than with HDL purified from control pregnant women (respectively 3.24 ± 1.25 μM versus 2.50 ± 0.95 μM, mean ± SD, P = 0.02). These data show that HDL from PE patients have less antioxidant effect than HDL from pregnant control women. S1P and apoM concentrations were significantly lower in HDL purified from PE women compared to HDL purified from control pregnant women, with a strong correlation ( r = 0.75) between the concentrations of these 2 parameters (respectively, 477 ± 131 versus 659 ± 120 nM/L for S1P and 1.5 ± 0.3 versus 2.1 ± 0.7 mg/dL for apoM, P < 0.05). These results show that HDL from PE patients contains less S1P and apoM than HDL from control pregnant women. We showed that HDL from PE pregnant women lose their antioxidant properties and are depleted in S1P and apoM, which could explain respectively the increase in LDL oxidation and the decrease in NO production by the endothelium previously described in the PE. These dysfunctional HDL, whose composition is modified, may be less protective for the vascular endothelium, a mechanism potentially involved in the pathophysiology of endothelial dysfunction seen in PE.

Apolipoprotein M: a novel adipokine decreasing with obesity and upregulated by calorie restriction

The adipose tissue (AT) is a secretory organ producing a wide variety of factors that participate in the genesis of metabolic disorders linked to excess fat mass. Weight loss improves obesity-related disorders. Transcriptomic studies on human AT, and a combination of analyses of transcriptome and proteome profiling of conditioned media from adipocytes and stromal cells isolated from human AT, have led to the identification of apolipoprotein M (apoM) as a putative adipokine. We aimed to validate apoM as novel adipokine, investigate the relation of AT APOM expression with metabolic syndrome and insulin sensitivity, and study the regulation of its expression in AT and secretion during calorie restriction-induced weight loss. We examined APOM mRNA level and secretion in AT from 485 individuals enrolled in 5 independent clinical trials, and in vitro in human multipotentadipose-derived stem cell adipocytes. APOM expression and secretion were measured during dieting. APOM was expressed in human subcutaneous and visceral AT, mainly by adipocytes. ApoM was released into circulation from AT, and plasma apoM concentrations correlate with AT APOM mRNA levels. In AT, APOM expression inversely correlated with adipocyte size, was lower in obese compared to lean individuals, and reduced in subjects with metabolic syndrome and type 2 diabetes. Regardless of fat depot, there was a positive relation between AT APOM expression and systemic insulin sensitivity, independently of fat mass and plasma HDL cholesterol. In human multipotentadipose-derived stem cell adipocytes, APOM expression was enhanced by insulin-sensitizing peroxisome proliferator-activated receptor agonists and inhibited by tumor necrosis factor α, a cytokine that causes insulin resistance. In obese individuals, calorie restriction increased AT APOM expression and secretion. ApoM is a novel adipokine, the expression of which is a hallmark of healthy AT and is upregulated by calorie restriction. AT apoM deserves further investigation as a potential biomarker of risk for diabetes and cardiovascular diseases.

Apolipoprotein status in type 2 diabetes mellitus and its complications (Review).

Dyslipidaemia in type 2 diabetes mellitus (T2DM) is characterized by high plasma triglyceride concentrations, reduced high‑density lipoprotein concentrations and increased small density low‑density lipoprotein concentrations. Dyslipidaemia may lead to cardiovascular disease (CVD) and other complications. Apolipoproteins mainly comprise six species, apolipoprotein (apo)A, apoB, apoC, apoD, apoE and apoM, which are important components of plasma lipoproteins that carry lipids and stabilize the structure of lipoproteins. Complex metabolic disorders of apolipoproteins are present in T2DM, such as high plasma apoB, apoC‑II, apoC‑III and apoE concentrations, and low plasma apoA‑I and apoM concentrations, which are associated with dyslipidaemia and interrelated complications. Plasma concentrations of some apolipoproteins are also altered in T2DM with CVD or other complications. Several apolipoprotein polymorphisms are associated with diabetes susceptibility and/or lipid metabolism. The present review described the metabolic disorders of apolipoproteins in T2DM and its complications, and the relationship between each major apolipoprotein and T2DM, as well as the effects of apolipoprotein polymorphisms on diabetic susceptibility.

Characteristics of lipid metabolism including serum apolipoprotein M levels in patients with primary nephrotic syndrome

BackgroundApolipoprotein M (apoM) is a 26-kD apolipoprotein that is mainly expressed in specific cell types, such as human liver parenchymal cells and kidney proximal renal tubular epithelial cells. ApoM can regulate the formation of pre-β-HDL and the reverse cholesterol transport and thus plays an important role in the metabolism of lipids and lipoproteins, meaning that it can affect the development of lipid metabolism disorders. Significantly elevated serum apoM levels are detected in patients with hyperlipidemia. However, few studies have shown how apoM is expressed in primary nephrotic syndrome (PNS), which is often accompanied with hyperlipidemia, and the underlying mechanism is poorly understood. This study was aimed at examining the apoM levels in patients with PNS and at determining the effects of PNS on serum apoM levels in these patients.MethodsThis study included patients with hyperlipidemia (n = 37), the PNS with hyperlipidemia group (n = 62), PNS without hyperlipidemia group (n = 33), and healthy controls (n = 73). The age and body–mass index (BMI) matched among the groups of participants. Their serum apoM concentrations were measured by an enzyme-linked immunosorbent assay. Serum levels of conventional lipids and renal function indices were assessed using an automatic biochemical analyzer. The data were analyzed by means of Pearson’s correlation coefficient (continuous variables) or Student’s t test (mean differences).ResultsThe average serum apoM concentrations were higher in the hyperlipidemia group (61.1 ± 23.2 mg/L, P = 0.004) than in the healthy controls (31.6 ± 18.92 mg/L). The serum apoM concentrations were lower in the PNS with hyperlipidemia group (25.1 ± 16.31 mg/L, P = 0.007) and in the PNS without hyperlipidemia group (21.00 ± 17.62 mg/L, P = 0.003) than in the healthy controls. The serum apoM concentrations in the PNS with hyperlipidemia group did not differ significantly from those in the PNS without hyperlipidemia group (P = 0.083). Moreover, serum apoM levels positively correlated with serum high-density lipoprotein cholesterol (HDL-C) and apoA1 levels and negatively correlated with proteinuria in PNS patients (r = 0.458, P = 0.003; r = 0.254, P = 0.022; r = −0.414, P = 0.028).ConclusionSerum apoM concentrations are higher in patients with hyperlipidemia than in healthy controls. Low serum apoM levels in patients with PNS are likely caused by PNS.

ApoM/HDL-C and apoM/apoA-I ratios are indicators of diabetic nephropathy in healthy controls and type 2 diabetes mellitus

BackgroundApolipoprotein M (apoM) concentrations were decreased in type 2 diabetes mellitus (T2DM). ApoM was selectively expressed in renal tubular epithelial cells. We investigated the changes in plasma apoM concentrations in diabetic nephropathy (DN) patients and the potential of apoM as a biomarker of DN. MethodsA total of 96 DN patients and 100 age- and sex-matched diabetic non-nephropathy (non-DN) patients and 110 healthy controls were included. All T2DM patients were divided into 3 groups according to urinary albumin excretion: normoalbuminuria (n=100), microalbuminuria (n=50) and macroalbuminuria (n=46). Plasma apoM concentrations were measured by enzyme-linked immunosorbent assay. ResultsDN Patients had higher plasma apoM concentrations than those in non-DN patients (22.23±11.69 vs. 18.96±7.85ng/μl, P<0.05). In addition, microalbuminuria group showed higher plasma apoM concentrations than those in normoalbuminuria group (22.67±11.40 vs. 18.96±7.85ng/μl, P<0.05). The areas under curve (AUC) of apoM using a receiver-operating characteristic (ROC) curve analysis showed that plasma apoM concentrations were not indicators for identification of DN from healthy people (AUC=0.478, P=0.585) and from T2DM (AUC=0.563, P=0.125). DN patients had higher ratios of apoM/HDL-C and apoM/apoA1 than those in healthy controls and in non-DN patients. ApoM/HDL-C and apoM/apoA1 ratios could be used as indicators for identification of DN from healthy people (AUC=0.597, P=0.016; AUC=0.665, P=0.000, respectively) and from T2DM (AUC=0.580, P=0.050; AUC=0.601, P=0.015, respectively). ConclusionsApoM/HDL-C and apoM/apoA1 ratios could be used as indicators for identification of DN from healthy people and from T2DM patients.

Vehicle-dependent Effects of Sphingosine 1-phosphate on Plasminogen Activator Inhibitor-1 Expression.

Aim: Sphingosine 1-phosphate (S1P) has been suggested to be a positive regulator of plasminogen activator inhibitor 1 (PAI-1) in adipocytes, while some studies are not consistent with this prothrombotic property of S1P. Since S1P is bound to apolipoprotein M (apoM) on HDL or to albumin in plasma, we compared the properties of these two forms on the PAI-1 induction.Methods: We investigated the associations of S1P, apoM, and PAI-1 concentrations in the plasma of normal coronary artery (NCA), stable angina pectoris (SAP), and acute coronary syndrome (ACS) subjects (n = 32, 71, and 38, respectively). Then, we compared the effects of S1P with various vehicles on the PAI-1 expression in 3T3L1 adipocytes. We also investigated the modulation of the PAI-1 levels in mice infected with adenovirus coding apoM.Results: Among ACS subjects, the PAI-1 level was positively correlated with the S1P level, but not the apoM level. In adipocytes, S1P bound to an apoM-rich vehicle induced PAI-1 expression to a lesser extent than the control vehicle, while S1P bound to an apoM-depleted vehicle induced PAI-1 expression to a greater extent than the control vehicle in 3T3L1 adipocytes. Additionally, apoM overexpression in mice failed to modulate the plasma PAI-1 level and the adipose PAI-1 expression level. S1P bound to albumin increased PAI-1 expression through the S1P receptor 2-Rho/ROCK-NFκB pathway.Conclusion: S1P bound to albumin, but not to apoM, induces PAI-1 expression in adipocytes, indicating that S1P can exert different properties on the pathogenesis of vascular diseases, depending on its vehicle.

Effects of hyperlipidaemia on plasma apolipoprotein M levels in patients with type 2 diabetes mellitus: an independent case-control study.

BackgroundApolipoprotein M (apoM) is mainly enriched in high-density lipoprotein (HDL) cholesterol and is slightly present in low-density lipoprotein (LDL) cholesterol and very low-density lipoprotein cholesterol. apoM is involved in HDL formation and HDL-mediated reverse cholesterol transport. apoM is also associated with hyperlipidaemia and type 2 diabetes mellitus (T2DM). Significantly high plasma apoM levels are detected in hyperlipidaemia mice with a defective LDL receptor. By contrast, low plasma apoM levels are observed in patients with T2DM, which is often accompanied with hyperlipidaemia. However, the underlying mechanism of this condition is poorly understood. This research aims to examine the changes in apoM levels in patients with hyperlipidaemia and to determine the effects of hyperlipidaemia on plasma apoM levels in patients with T2DM.MethodsThis study included patients with hyperlipidaemia (n = 79), patients with T2DM but without hyperlipidaemia (n = 125), patients with T2DM and hyperlipidaemia (n = 98), and healthy controls (n = 105). Their plasma apoM concentrations were measured with enzyme-linked immunosorbent assay.ResultsThe average plasma apoM concentrations were 18 % higher in the hyperlipidaemia group (26.63 ± 10.35 ng/μL) than in the healthy controls (22.61 ± 10.81 ng/μL, P <0.01). The plasma apoM concentrations were lower in the T2DM without hyperlipidaemia group (18.54 ± 10.33 ng/μL, P <0.01) and the T2DM with hyperlipidaemia group (19.83 ± 7.41 ng/μL, P <0.05) than in the healthy controls. Similar to apoA-I (1.29 ± 0.33 g/L vs. 1.28 ± 0.31 g/L, P >0.05), the plasma apoM concentrations in the T2DM with hyperlipidaemia group did not significantly differ from those in the T2DM without hyperlipidaemia group (P >0.05). Multivariate linear regression analysis showed that hyperlipidaemia (β = 5.18, P = 0.007) is an independent promoting factor of plasma apoM levels and diabetes (β = −3.09, P = 0.005) is an inhibiting factor of plasma apoM levels.ConclusionPlasma apoM concentrations are higher in patients with hyperlipidaemia than in healthy controls. Low plasma apoM levels in patients with T2DM are likely caused by diabetes but are not induced by hyperlipidaemia.

Correlation analysis between ApoM gene-promoter polymorphisms and coronary heart disease.

SummaryObjectives:Apolipoprotein M (ApoM), a 25-kDa plasma protein belonging to the lipocalin protein family, is predominantly associated with high-density lipoprotein cholesterol (HDL-C). Studies have suggested ApoM to be important for the formation of pre-β-HDL and to increase cholesterol efflux from macrophage foam cells. The aim of this study was to explore the association of single-nucleotide polymorphisms(SNPs) in the ApoM promoter with coronary atherosclerotic disease (CAD), and the contribution of latent factors.Methods:ApoM was measured in samples from two separate case–control studies, of whom 88 patients developed CAD and 88 were controls. Whole-blood samples from subjects were genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP). Luciferase activities were measured for HepG2 cells with two SNPs, rs805296 (T-778C) and rs940494 (T-855C), and after interfering with or overexpressing the predicted transcription factors. The ability of the SNPs to combine with nucleoproteins was analysed by electophoretic mobility shift assay (EMSA).Results:Mean plasma ApoM concentrations in the CAD and non-CAD groups were 9.58 ± 4.30 and 12.22 ± 6.59 μg/ ml, respectively. Correlation studies of ApoM concentrations with several analytes showed a marked positive correlation with HDL-C, fasting plasma glucose and triglyceride levels. The CC genotype showed lower luciferase activities compared to the TC and TT genotypes. The ApoM-855 mutant-typecould bind to the AP-2α. Interference and overexpression of AP-2 increased and decreased luciferase activities of the wild and mutant types to different degrees.Conclusion::ApoM may be a biomarker of CAD. ApoM- 855 T→C substitution provides binding sites for AP-2α and reduces ApoM transcription activity.

A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive signaling lysophospholipids that activate specific G protein-coupled receptors on the cell surface triggering numerous biological events. In circulation, S1P and LPA associate with specific carrier proteins or chaperones; serum albumin binds both S1P and LPA while HDL shuttles S1P via interactions with apoM. We used a series of kinetic exclusion assays in which monoclonal anti-S1P and anti-LPA antibodies competed with carrier protein for the lysophospholipid to measure the equilibrium dissociation constants (Kd) for these carrier proteins binding S1P and the major LPA species. Fatty acid-free (FAF)-BSA binds these lysophospholipids with the following Kd values: LPA(16:0), 68 nM; LPA(18:1), 130 nM; LPA(18:2), 350 nM; LPA(20:4), 2.2 μM; and S1P, 41 μM. FAF human serum albumin binds each lysophospholipid with comparable affinities. By measuring the apoM concentration and expanding the model to include endogenous ligand, we were able to resolve the Kd values for S1P binding apoM in the context of human HDL and LDL particles (21 nM and 2.4 nM, respectively). The novel competitive assay and analysis described herein enables measurement of Kd values of completely unmodified lysophospholipids binding unmodified carrier proteins in solution, and thus provide insights into S1P and LPA storage in the circulation system and may be useful in understanding chaperone-dependent receptor activation and signaling.

A single-nucleotide polymorphism C-724 /del in the proter region of the apolipoprotein M gene is associated with type 2 diabetes mellitus.

BackgroundApolipoprotein M (apoM) was the carrier of the biologically active lipid mediator sphingosine-1-phospate in high density lipoprotein cholesterol (HDL-C) and played a critical role in formation and maturation of prebeta-HDL-C particles. The plasma apoM levels were decreased obviously in patients with type 2 diabetes mellitus (T2DM). A new single-nucleotide polymorphism (SNP) C-724del in apoM promoter was associated with a higher risk for coronary artery diseases (CAD) and myocardial infarction, could reduce promoter activities and apoM expression in vitro. The primary aim of the present case-controls study was to investigate the effect of apoM SNP C-724del on apoM expression in vivo and its association with T2DM susceptibility in an eastern Han Chinese cohort.MethodsTwo hundred and fifty-nine T2DM patients and seventy-six healthy controls were included in this study. Amplifying DNA of apoM proximal promoter region including SNP C-724del by Real-Time Polymerase Chain Reaction (RT-PCR) and amplicons sequencing. The plasma apoM concentrations were assayed by enzyme linked immunosorbentassay (ELISA).ResultsFour polymorphic sites, rs805297 (C-1065A), rs9404941 (T-855C), rs805296 (T-778C), C-724del were confirmed. rs805297 (C-1065A) and rs9404941 (T-855C) showed no statistical difference in allele frequencies and genotype distributions between T2DM patients and healthy controls just as previous studies. It’s worth noting that the difference of rs805296 (T-778C) between these two groups was not found in this study. In SNP C-724del, the frequency of del allele and mutant genotypes (del/del, C/del) were higher in T2DM patients compared with healthy controls (p = 0.035; P = 0.040, respectively), while the plasma apoM levels of C-724del mutant allele carriers compared with the wide-type homozygotes carriers were not statistically different in T2DM patients (18.20 ± 8.53 ng/uL vs 20.44 ± 10.21 ng/uL, P = 0.245).ConclusionThe polymorphism C-724del in the promoter region of the apoM gene could confer the risk of T2DM among eastern Han Chinese. Unfortunately, the lowing of plasma apoM levels of C-724del mutant allele carriers compared with the wide-type homozygotes carriers in T2DM patients was not statistically different in present study, so further researchs were needed by enlarging the sample.

Urinary Apolipoprotein M Could Be Used as a Biomarker of Acute Renal Injury: An Ischemia-Reperfusion Injury Model of Kidney in Rat

BackgroundIt has been well documented that apolipoprotein M (apoM) is principally expressed in hepatocytes as well as renal tubular epithelial cells. The importance of apoM in the kidney is unknown. In the present study we examined urinary any apoM after short-term ischemia-reperfusion injury (IRI) of kidney in a rat model. MethodsThe kidneys of 11 male Sprague-Dawley rats were rendered ischemic for 45 minutes followed by different intervals of reperfusion. Serum and urine apoM concentrations were determined using a dot-blot analysis with specific rabbit anti-human apoM antibodies that cross-react with rat apoM. Serum concentrations of blood urea nitrogen (BUN) and creatinine (Cr) were determined using standard clinical automated analyses. ResultsBUN was significantly elevated after 45 minutes of ischemia followed by 24 hours of reperfusion; serum Cr concentrations were also significantly increased at 6 and 24 hours of reperfusion. Interestingly, similar to BUN and Cr, serum apoM concentrations were significantly increased after ischemia for 45 minutes alone and after 2 hours of reperfusion. Urinary apoM concentrations were obviously increased after 2 h as well as 6 hours of reperfusion. ConclusionapoM showed characteristics of an acute-phase reactive protein; its occurrence in urine may be considered to be a biomarker of acute renal injury.

Apolipoprotein M can discriminate HNF1A‐MODY from Type 1 diabetes

Missed diagnosis of maturity-onset diabetes of the young (MODY) has led to an interest in biomarkers that enable efficient prioritization of patients for definitive molecular testing. ApolipoproteinM (apoM) was suggested as a biomarker for hepatocyte nuclear factor1 alpha (HNF1A)-MODY because of its reduced expression in Hnf1a(-/-) mice. However, subsequent human studies examining apoM as a biomarker have yielded conflicting results. We aimed to evaluate apoM as a biomarker for HNF1A-MODY using a highly specific and sensitive ELISA. ApoM concentration was measured in subjects with HNF1A-MODY (n=69), Type1 diabetes (n=50), Type2 diabetes (n=120) and healthy control subjects (n=100). The discriminative accuracy of apoM and of the apoM/HDL ratio for diabetes aetiology was evaluated. Mean (standard deviation) serum apoM concentration (μmol/l) was significantly lower for subjects with HNF1A-MODY [0.86 (0.29)], than for those with Type1 diabetes [1.37 (0.26), P=3.1×10(-18) ) and control subjects [1.34 (0.22), P=7.2×10(-19) ). There was no significant difference in apoM concentration between subjects with HNF1A-MODY and Type2 diabetes [0.89 (0.28), P=0.13]. The C-statistic measure of discriminative accuracy for apoM was 0.91 for HNF1A-MODY vs. Type1 diabetes, indicating high discriminative accuracy. The apoM/HDL ratio was significantly lower in HNF1A-MODY than other study groups. However, this ratio did not perform well in discriminating HNF1A-MODY from either Type1 diabetes (C-statistic=0.79) or Type2 diabetes (C-statistic=0.68). We confirm an earlier report that serum apoM levels are lower in HNF1A-MODY than in controls. Serum apoM provides good discrimination between HNF1A-MODY and Type1 diabetes and warrants further investigation for clinical utility in diabetes diagnostics.

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.

Vascular Endothelial Growth Factor Downregulates Apolipoprotein M Expression by Inhibiting Foxa2 in a Nur77-Dependent Manner

We aimed to investigate whether vascular endothelial growth factor (VEGF) influences apolipoprotein M (ApoM) expression and pre-β-high-density lipoprotin (HDL) formation, and whether forkhead box A2 (Foxa2) and Nur77 are involved in this process. We analyzed the serum VEGF concentrations of 264 adults who underwent a medical checkup and found that VEGF concentration was positively correlated with serum triglyceride, total cholesterol, LDL cholesterol (LDL-C), very-low-density lipoprotein cholesterol (VLDL-C), and ApoB concentrations, but was negatively correlated with serum high-density lipoprotein cholesterol (HDL-C) and ApoM concentrations. We further investigated the effects of VEGF on ApoM expression and pre-β-HDL formation, and the mechanisms responsible, in HepG2 cells and mouse primary hepatocytes. VEGF markedly downregulated ApoM expression and pre-β-HDL formation. At the same time, expression of Foxa2 was also inhibited, whereas expression of Nur77 was increased by treatment with VEGF. Furthermore, small interfering (si) RNA knockdown of Foxa2 made the downregulation of VEGF on ApoM expression and pre-β-HDL formation even more obvious. In addition, siRNA knockdown of Nur77 significantly compensated for the inhibitory effect of VEGF on Foxa2 expression, whereas the Nur77 agonist cytosporone B led to the downregulation of Foxa2 expression more significantly than VEGF. Moreover, overexpression of a Nur77 transgene in C57BL/6 mice resulted in decreased serum ApoM and pre-β-HDL levels, whereas si-Nur77-treated mice displayed upregulated serum ApoM and pre-β-HDL levels. These results provide evidence that VEGF may first downregulate expression of Foxa2 by enhancing Nur77 activity and then decrease expression of ApoM and pre-β-HDL formation. Therefore, our study may be useful in understanding the critical effect of VEGF in the pathogenesis of atherosclerosis.

Reduced expression and secretion of apolipoprotein M in fat-fed, streptozotocin-diabetic rats is partially reversed by an artificial ligand of PPARγ

To investigate the effect of administration of rosiglitazone, an artificial ligand of PPARγ, on the expression and secretion of apolipoprotein (apoM) in fat-fed, streptozotocin-treated rats, an animal model for type 2-like diabetes. Healthy male SD rats were divided into 4 groups: a control group (n=7), a high-fat chow group (HF group, n=8), a diabetes mellitus group (DM group, n=7), and a diabetes mellitus group with rosiglitazone intervention group (RSG group, n=7). Fasting blood glucose (FBG), fasting insulin (FINS), triglyceride (TG) and total cholesterol (TC) were measured at the beginning of the study. The diabetic rats model was established by feeding high fat chow and intraperitoneal injection of streprozotocin. Then the randomly selected treatment group was given rosiglitazone by daily gavage for 8 weeks. All the rats were killed at the fifteenth week, at which time blood and tissues (liver, kidney, adipose) were collected and prepared. The levels of FBG, FINS, TG and TC were assayed. The level of apoM in serum was measured by enzyme-linked immunosorbent assay (ELISA). Reverse transcription polymerase chain reaction (RT-PCR) was used to determine apoM mRNA expression in liver, kidney, and adipose tissues. Compared with either control group or HF group, serum apoM concentration in the DM group was reduced significantly (P<0.05); compared with the DM group, however, serum apoM concentrations in RSG group were increased (P<0.05). The expression of apoM mRNA in liver was highest, in kidney medium, and in adipose tissue extremely low (P<0.05). ApoM mRNA expression in liver and kidney was decreased in both DM and HF groups compared to control group (P<0.05). But, as with serum apoM concentration, apoM mRNA in the liver, kidney and adipose tissues of the RSG group were all increased markedly (P<0.05). The level of serum apoM in SD rats correlated negatively with TG (r=-0.466, P=0.011), TC (r=-0.568, P= 0.001), FBS (r =-0.371, P<0.001), and FINS(r=-0.768, P= 0.048 ). These results suggest that apoM may participate in the glucose and lipid metabolism by the regulation of PPARγ.