Makoto Kurano

Liver involvement in sphingosine 1-phosphate dynamism revealed by adenoviral hepatic overexpression of apolipoprotein M

OBJECTIVES Sphingosine 1-phosphate (S1P) is a vasoprotective lipid mediator that is mainly carried on HDL in the circulation and several anti-atherosclerotic properties of HDL is considered to be ascribed to S1P. Since S1P riding on HDL was recently shown to bind to apolipoprotein M (apoM), which is derived from liver, we analyzed the possible involvement of liver in S1P metabolism. METHODS AND RESULTS Using adenoviruses, we overexpressed apoM in HepG2 cells and mice livers and found that both the medium/plasma and cell/liver S1P contents increased. Among lipoprotein subclasses, S1P contents increased mainly in HDL fractions. On the other hand, hepatectomy resulted in the reduction of plasma S1P levels in mice. The incubation of S1P in the conditional medium of apoM-overexpressing HepG2 cells interfered with S1P degradation. Furthermore, adenoviral hepatic overexpression of apoM resulted in increase in the S1P level of plasma but not of blood cells, while combination of hepatic apoM overexpression and intraperitoneal administration of C₁₇-sphingosine resulted in the increase in the C₁₇-S1P level both in livers and in plasma, but again not in blood cells. CONCLUSIONS Livers are involved in S1P dynamism, and it was suggested that apoM, produced from livers, increases circulating plasma S1P by augmenting the S1P output from livers and modifies extracellular S1P metabolism.

Possible Involvement of Minor Lysophospholipids in the Increase in Plasma Lysophosphatidic Acid in Acute Coronary Syndrome

Objective—Lysophosphatidic acids (LPA) have important roles in the field of vascular biology and are derived mainly from lysophosphatidylcholine via autotaxin. However, in our previous study, only the plasma LPA levels, and not the serum autotaxin levels, increased in patients with acute coronary syndrome (ACS). The aim of this study was to elucidate the pathway by which LPA is increased in patients with ACS. Approach and Results—We measured the plasma lysophospholipids species in 141 consecutive patients undergoing coronary angiography (ACS, n=38; stable angina pectoris, n=71; angiographically normal coronary arteries, n=32) using a liquid chromatography-tandem mass spectrometry analysis. Among the ACS subjects, notable increases in the 22:6 LPA, 18:2 LPA, and 20:4 LPA levels were observed. The in vitro experiments revealed that serum incubation mainly increased the 18:2 LPA level, whereas platelet activation increased the 20:4 LPA level. Minor lysophospholipids other than LPA were also elevated in ACS subjects and were well correlated with the corresponding LPA species, including 22:6 LPA. A multiple regression analysis also revealed that lysophosphatidylinositol, lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylglycerol were independent explanatory variables for several LPA species. Conclusions—Specific LPA species, especially long-chain unsaturated LPA, were elevated in ACS patients, along with the corresponding minor lysophospholipids. The elevation of these LPA species might be mainly caused by presently unidentified LPA-producing pathway(s). Minor lysophospholipids might be involved in the generation of LPA, especially 22:6 LPA, and in the pathogenesis of ACS.

Sphingosine 1-phosphate release from platelets during clot formation: close correlation between platelet count and serum sphingosine 1-phosphate concentration

BackgroundSphingosine 1-phosphate (Sph-1-P), abundantly stored in platelets and released extracellularly upon activation, plays important roles as an extracellular mediator by interacting with specific cell surface receptors, especially in the area of vascular biology and immunology/hematology. Although the plasma Sph-1-P level is reportedly determined by red blood cells (RBCs), but not platelets, this may not be true in cases where the platelets have been substantially activated.Methods and resultsWe measured the Sph-1-P and dihydrosphingosine 1-phosphate (DHSph-1-P) levels in serum samples (in which the platelets had been fully activated) from subjects with (n = 21) and without (n = 33) hematological disorders. We found that patients with essential thrombocythemia exhibited higher serum Sph-1-P and DHSph-1-P concentrations. The serum Sph-1-P concentration was closely correlated with the platelet count but was very weakly correlated with the RBC count. Similar results were obtained for DHSph-1-P. The serum Sph-1-P and DHSph-1-P levels were inversely correlated with the level of autotaxin (ATX), a lysophosphatidic acid-producing enzyme. A multiple regression analysis also revealed that the platelet count had the greatest explanatory impact on the serum Sph-1-P level.ConclusionsOur present results showed close correlations between both the serum Sph-1-P and DHSph-1-P levels and the platelet count (but not the RBC count); these results suggest that high concentrations of these sphingoid base phosphates may be released from platelets and may mediate cross talk between platelet activation and the formation of atherosclerotic lesions.

Increased lysophosphatidic acid levels in culprit coronary arteries of patients with acute coronary syndrome

BACKGROUND Lysophosphatidic acid (LPA) is a platelet activator and highly thrombogenic lipid constituent of atherosclerotic plaque. However, whether or not LPA locally released from culprit lesions is associated with acute coronary syndrome (ACS) remains unclear. METHODS We studied 52 patients with ACS who were treated by emergency percutaneous coronary intervention and thrombectomy. Levels of LPA and other established biomarkers were enzymatically assayed in samples of culprit coronary arterial and systemic peripheral arterial blood. Levels of LPA and lysophosphatidylcholine (LPC) were measured in plasma, and those of autotaxin, soluble CD40 ligand (sCD40L), hs-CRP and Lp-PLA2 were measured in serum. RESULTS Median LPA levels were significantly higher in coronary (CB) than in peripheral (PB) arterial blood (p = 0.009). Levels of sCD40L were higher in CB than in PB, but the difference did not reach statistical significance (p = 0.177). In contrast, autotaxin and Lp-PLA2 levels were significantly higher in PB than in CB (p = 0.005 and p = 0.038, respectively). Levels of LPC and hs-CRP were also higher in PB than in CB (p = 0.129 and p = 0.121, respectively). Levels of LPA in both CB and PB were positively and significantly associated with those of LPC (r = 0.632, p < 0.01 and r = 0.465, p < 0.001). CONCLUSIONS Culprit coronary arteries of ACS contained significantly more LPA than the systemic arterial circulation. Higher LPA concentrations might be associated with the pathophysiology of ACS.

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.

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

Background: A positive correlation exists between sphingosine 1-phosphate (S1P) and LDL cholesterol. Results: Hepatic LDL receptor overexpression decreased plasma S1P together with apoM in wild-type mice, but not in apoE-deficient mice. Conclusion: LDL receptor is involved in the clearance of S1P, utilizing apoE as a ligand. Significance: We propose the novel role of LDL receptor and apoE in the clearance of S1P. 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.

Modulation of sphingosine-1-phosphate and apolipoprotein M levels in the plasma, liver and kidneys in streptozotocin-induced diabetic mice.

Sphingosine‐1‐phosphate (S1P), a multifunctional bioactive lipid mediator, is involved in various diseases. Apolipoprotein M (ApoM) carries S1P on high‐density lipoprotein and modulates S1P metabolism to increase the total S1P mass in the body. Both S1P and ApoM are involved in diabetes.

Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.

LXR agonist increases apoE secretion from HepG2 spheroid, together with an increased production of VLDL and apoE-rich large HDL

BackgroundThe physiological regulation of hepatic apoE gene has not been clarified, although the expression of apoE in adipocytes and macrophages has been known to be regulated by LXR.Methods and ResultsWe investigated the effect of TO901317, a LXR agonist, on hepatic apoE production utilizing HepG2 cells cultured in spheroid form, known to be more differentiated than HepG2 cells in monolayer culture. Spheroid HepG2 cells were prepared in alginate-beads. The secretions of albumin, apoE and apoA-I from spheroid HepG2 cells were significantly increased compared to those from monolayer HepG2 cells, and these increases were accompanied by increased mRNA levels of apoE and apoA-I. Several nuclear receptors including LXRα also became abundant in nuclear fractions in spheroid HepG2 cells. Treatment with TO901317 significantly increased apoE protein secretion from spheroid HepG2 cells, which was also associated with the increased expression of apoE mRNA. Separation of the media with FPLC revealed that the production of apoE-rich large HDL particles were enhanced even at low concentration of TO901317, and at higher concentration of TO901317, production of VLDL particles increased as well.ConclusionsLXR activation enhanced the expression of hepatic apoE, together with the alteration of lipoprotein particles produced from the differentiated hepatocyte-derived cells. HepG2 spheroids might serve as a good model of well-differentiated human hepatocytes for future investigations of hepatic lipid metabolism.

Sphingosine kinase-1, S1P transporter spinster homolog 2 and S1P2 mRNA expressions are increased in liver with advanced fibrosis in human

The role of sphingosine 1-phosphate (S1P) in liver fibrosis or inflammation was not fully examined in human. Controversy exists which S1P receptors, S1P1 and S1P3 vs S1P2, would be importantly involved in its mechanism. To clarify these matters, 80 patients who received liver resection for hepatocellular carcinoma and 9 patients for metastatic liver tumor were enrolled. S1P metabolism was analyzed in background, non-tumorous liver tissue. mRNA levels of sphingosine kinase 1 (SK1) but not SK2 were increased in livers with fibrosis stages 3–4 compared to those with 0–2 and to normal liver. However, S1P was not increased in advanced fibrotic liver, where mRNA levels of S1P transporter spinster homolog 2 (SPNS2) but not S1P-degrading enzymes were enhanced. Furthermore, mRNA levels of S1P2 but not S1P1 or S1P3 were increased in advanced fibrotic liver. These increased mRNA levels of SK1, SPNS2 and S1P2 in fibrotic liver were correlated with α-smooth muscle actin mRNA levels in liver, and with serum ALT levels. In conclusion, S1P may be actively generated, transported to outside the cells, and bind to its specific receptor in human liver to play a role in fibrosis or inflammation. Altered S1P metabolism in fibrotic liver may be their therapeutic target.