We used a HPLC-MS/MS methodology for determination of a basic metabolomic profile (18:1,18:0 sphingoid backbone, C(14)-C(26) N-acyl part) of "normal" sphingolipid levels in human serum and plasma. Blood was collected from healthy males and nonpregnant females under fasting and nonfasting conditions with and without anticoagulants. Sphingolipids analyzed included sphingoid bases, sphingosine and dihydrosphingosine, their 1-phosphates (S1P and dhS1P), molecular species (C(n)-) of ceramide (Cer), sphingomyelin (SM), hexosylceramide (HexCer), lactosylceramide (LacCer), and Cer 1-phosphate (Cer1P). SM, LacCer, HexCer, Cer, and Cer1P constituted 87.7, 5.8, 3.4, 2.8, and 0.15% of total sphingolipids, respectively. The abundant circulating SM was C(16)-SM (64.0 µM), and it increased with fasting (100 µM). The abundant LacCer was C(16)-LacCer (10.0 µM) and the abundant HexCer was C(24)-HexCer (2.5 µM). The abundant Cer, C(24)-Cer (4.0 µM), was not influenced by fasting; however, levels of C(16)-C(20) Cers were decreased in response to fasting. S1P levels were higher in serum than plasma (0.68 µM vs. 0.32 µM). We also determined levels of sphingoid bases and SM species in isolated lipoprotein classes. HDL(3) was the major carrier of S1P, dhS1P, and Sph, and LDL was the major carrier of Cer and dhSph. Per particle, VLDL contained the highest levels of SM, Cer, and S1P. HPLC-MS/MS should provide a tool for clinical testing of circulating bioactive sphingolipids in human blood.

Apolipoprotein J (apoJ) has been shown to be the predominant amyloid beta-peptide (Abeta)-binding protein in cerebrospinal fluid. We have previously demonstrated that the endocytic receptor low density lipoprotein receptor-related protein-2/megalin (LRP-2), which is expressed by choroid plexus epithelium and ependymal cells lining the brain ventricles and neural tube, binds and mediates cellular uptake of apoJ (Kounnas, M. Z., Loukinova, E. B., Stefansson, S., Harmony, J. A., Brewer, B., Strickland, D. K., and Argraves, W. S. (1995) J. Biol. Chem. 270, 13070-13075). In the present study, we evaluated the ability of apoJ to mediate binding of Abeta1-40-apoJ complex to LRP-2 in vitro. Immunoblot analysis showed that incubation of apoJ with Abeta1-40 resulted in the formation of Abeta1-40-apoJ complex and the inhibition of the formation of Abeta1-40 aggregates. Using an enzyme-linked immunosorbent assay, an estimated dissociation constant (Kd) of 4.8 nM was derived for the interaction between Abeta1-40 and apoJ. Enzyme-linked immunosorbent assay was also used to study the interaction of the Abeta1-40-apoJ complex with LRP-2. The results showed that Abeta alone did not bind directly to LRP-2; however, when Abeta1-40 was combined with apoJ to form a complex, binding to LRP-2 took place. The binding interaction could be blocked by inclusion of the receptor-associated protein, an antagonist of apoJ binding to LRP-2. When LRP-2-expressing cells were given 125I-Abeta1-40, cellular uptake of the radiolabeled peptide was promoted by co-incubation with apoJ. When the cells were provided purified 125I-Abeta1-40-apoJ complex, the complex was internalized and degraded, and both processes were inhibited with polyclonal LRP-2 antibodies. Furthermore, chloroquine treatment inhibited the cellular degradation of the complex. The data indicate that apoJ facilitates Abeta1-40 binding to LRP-2 and that the receptor mediates cellular clearance of Abeta1-40-apoJ complex leading to lysosomal degradation of Abeta1-40. The findings support the possibility that LRP-2 can act in vivo to mediate clearance of the complex from biological fluids such as cerebrospinal fluid and thereby play a role in the regulation of Abeta accumulation.

Receptors that endocytose high-density lipoproteins (HDL) have been elusive. Here yolk-sac endoderm-like cells were used to identify an endocytic receptor for HDL. The receptor was isolated by HDL affinity chromatography and identified as cubilin, the recently described endocytic receptor for intrinsic factor-vitamin B ₁₂ . Cubilin antibodies inhibit HDL endocytosis by the endoderm-like cells and in mouse embryo yolk-sac endoderm, a prominent site of cubilin expression. Cubilin-mediated HDL endocytosis is inhibitable by HDL ₂ , HDL ₃ , apolipoprotein (apo)A-I, apoA-II, apoE, and RAP, but not by low-density lipoprotein (LDL), oxidized LDL, VLDL, apoC-I, apoC-III, or heparin. These findings, coupled with the fact that cubilin is expressed in kidney proximal tubules, suggest a role for this receptor in embryonic acquisition of maternal HDL and renal catabolism of filterable forms of HDL.

Cubilin has recently been shown to function as an endocytic receptor for high density lipoproteins (HDL). The lack of apparent transmembrane and cytoplasmic domains in cubilin raises questions as to the means by which it can mediate endocytosis. Since cubilin has been reported to bind the endocytic receptor megalin, we explored the possibility that megalin acts in conjunction with cubilin to mediate HDL endocytosis. While megalin did not bind to HDL, delipidated HDL, or apoA-I, it was found to copurify with cubilin isolated by HDL-Sepharose affinity chromatography. Cubilin and megalin exhibited coincident patterns of mRNA expression in mouse tissues including the kidney, ileum, thymus, placenta, and yolk sac endoderm. The expression of both receptors in yolk sac endoderm-like cells was inducible by retinoic acid treatment but not by conditions of sterol depletion. Suppression of megalin activity or expression by treatment with either megalin antibodies or megalin antisense oligodeoxynucleotides resulted in inhibition of cubilin-mediated endocytosis of HDL. Furthermore, megalin antisense oligodeoxynucleotide treatment resulted in reduced cell surface expression of cubilin. These data demonstrate that megalin acts together with cubilin to mediate HDL endocytosis and further suggest that megalin may play a role in the intracellular trafficking of cubilin.

Aims/hypothesis In previous studies we have shown that extravasated, modified LDL is associated with pericyte loss, an early feature of diabetic retinopathy (DR). Here we sought to determine detailed mechanisms of this LDL-induced pericyte loss. Methods Human retinal capillary pericytes (HRCP) were exposed to ‘highly-oxidised glycated’ LDL (HOG-LDL) (a model of extravasated and modified LDL) and to 4-hydroxynonenal or 7-ketocholesterol (components of oxidised LDL), or to native LDL for 1 to 24 h with or without 1 h of pretreatment with inhibitors of the following: (1) the scavenger receptor (polyinosinic acid); (2) oxidative stress (N-acetyl cysteine); (3) endoplasmic reticulum (ER) stress (4-phenyl butyric acid); and (4) mitochondrial dysfunction (cyclosporin A). Oxidative stress, ER stress, mitochondrial dysfunction, apoptosis and autophagy were assessed using techniques including western blotting, immunofluorescence, RT-PCR, flow cytometry and TUNEL assay. To assess the relevance of the results in vivo, immunohistochemistry was used to detect the ER stress chaperon, 78 kDa glucose-regulated protein, and the ER sensor, activating transcription factor 6, in retinas from a mouse model of DR that mimics exposure of the retina to elevated glucose and elevated LDL levels, and in retinas from human participants with and without diabetes and DR. Results Compared with native LDL, HOG-LDL activated oxidative and ER stress in HRCP, resulting in mitochondrial dysfunction, apoptosis and autophagy. In a mouse model of diabetes and hyperlipidaemia (vs mouse models of either condition alone), retinal ER stress was enhanced. ER stress was also enhanced in diabetic human retina and correlated with the severity of DR. Conclusions/interpretation Cell culture, animal, and human data suggest that oxidative stress and ER stress are induced by modified LDL, and are implicated in pericyte loss in DR.