Adipocyte differentiation-related protein is induced by LRP1-mediated aggregated LDL internalization in human vascular smooth muscle cells and macrophages

Aggregated LDL (agLDL) is internalized by LDL receptor-related protein (LRP1) in vascular smooth muscle cells (VSMCs) and human monocyte-derived macrophages (HMDMs). AgLDL is, therefore, a potent inducer of massive intracellular cholesteryl ester accumulation in lipid droplets. The adipocyte differentiation-related protein (ADRP) has been found on the surface of lipid droplets. The objectives of this work were to analyze whether agLDL uptake modulates ADRP expression levels and whether the effect of agLDL internalization on ADRP expression depends on LRP1 in human VSMCs and HMDMs. AgLDL strongly upregulates ADRP mRNA (real-time PCR) and protein expression (Western blot) in human VSMCs (mRNA: by 3.06-fold; protein: 8.58-fold) and HMDMs (mRNA: by 3.5-fold; protein: by 3.71-fold). Treatment of VSMCs and HMDMs with small anti-LRP1-interfering RNA (siRNA-LRP1) leads to specific inhibition of LRP1 expression. siRNA-LRP1 treatment significantly reduced agLDL-induced ADRP overexpression in HMDMs (by 69%) and in VSMCs (by 53%). Immunohystochemical studies evidence a colocolocalization between ADRP/macrophages and ADRP/VSMCs in advanced lipid-enriched atherosclerotic plaques. These results demonstrate that agLDL-LRP1 engagement induces ADRP overexpression in both HMDMs and human VSMCs and that ADRP is highly expressed in advanced lipid-enriched human atherosclerotic plaques. Therefore, LRP1-mediated agLDL uptake might play a pivotal role in vascular foam cell formation.     

Evaluation of the causality of the low‐density lipoprotein receptor gene (LDLR) for serum lipids in pigs

A significant quantitative trait locus (QTL) for low‐density lipoprotein cholesterol (LDL‐C) and total cholesterol (TC) was identified around the LDLR gene on chromosome 2 (SSC2) in a White Duroc × Erhualian F₂ resource population and Sutai pigs in our previous study. However, in previous reports, the causality of LDLR with serum lipids is controversial in pigs. To systematically assess the causality of LDLR with serum lipids, association analyses were successively performed in three populations: Sutai pigs, a White Duroc × Erhualian F₂ resource population and a Duroc × (Landrace × Large White) population. We first performed a haplotype‐based association study with 60K SNP genotyping data and evidenced the significant association with LDL‐C and TC around the LDLR gene region. We also found that there is more than one QTL for LDL‐C and TC on SSC2. Then, we evaluated the causalities of two missense mutations, c.1812C>T and c.1520A>G, with LDL‐C and TC. We revealed that the c.1812C>T SNP showed the strongest association with LDL‐C (P = 5.40 × 10^?11) and TC (P = 3.64 × 10^?8) and explained all the QTL effect in Sutai pigs. Haplotype analysis found that two missense SNPs locate within a 1.93‐Mb haplotype block. One major haplotype showed the strongest significant association with LDL‐C (P = 4.62 × 10^?18) and TC (P = 1.06 × 10^?9). However, the c.1812C>T SNP was not identified in the White Duroc × Erhualian intercross, and the association of c.1520A>G with both LDL‐C and TC did not achieve significance in this F₂ population, suggesting population heterogeneity. Both missense mutations were identified in the Duroc × (Landrace × Large White) population and showed significant associations with LDL‐C and TC. Our data give evidence that the LDLR gene should be a candidate causative gene for LDL‐C and TC in pigs, but heterogeneity exists in different populations.     

High density lipoprotein metabolism in low density lipoprotein receptor-deficient mice

The LDL receptor (LDLR) and scavenger receptor class B type I (SR-BI) play physiological roles in LDL and HDL metabolism in vivo. In this study, we explored HDL metabolism in LDLR-deficient mice in comparison with WT littermates. Murine HDL was radiolabeled in the protein (¹²⁵I) and in the cholesteryl ester (CE) moiety ([³H]). The metabolism of ¹²⁵I-/[³H]HDL was investigated in plasma and in tissues of mice and in murine hepatocytes. In WT mice, liver and adrenals selectively take up HDL-associated CE ([³H]). In contrast, in LDLR^−/− mice, selective HDL CE uptake is significantly reduced in liver and adrenals. In hepatocytes isolated from LDLR^−/− mice, selective HDL CE uptake is substantially diminished compared with WT liver cells. Hepatic and adrenal protein expression of lipoprotein receptors SR-BI, cluster of differentiation 36 (CD36), and LDL receptor-related protein 1 (LRP1) was analyzed by immunoblots. The respective protein levels were identical both in hepatic and adrenal membranes prepared from WT or from LDLR^−/− mice. In summary, an LDLR deficiency substantially decreases selective HDL CE uptake by liver and adrenals. This decrease is independent from regulation of receptor proteins like SR-BI, CD36, and LRP1. Thus, LDLR expression has a substantial impact on both HDL and LDL metabolism in mice.     

The expression of MMP‐1 and MMP‐9 is up‐regulated by smooth muscle cells after their cross‐talk with macrophages in high glucose conditions

Patients with diabetes mellitus have an increased risk of myocardial infarction and coronary artery disease‐related death, exhibiting highly vulnerable plaques. Many studies have highlighted the major role of macrophages (MAC) and smooth muscle cells (SMC) and the essential part of metalloproteases (MMPs) in atherosclerotic plaque vulnerability. We hypothesize that in diabetes, the interplay between MAC and SMC in high glucose conditions may modify the expression of MMPs involved in plaque vulnerability. The SMC‐MAC cross‐talk was achieved using trans‐well chambers, where human SMC were grown at the bottom and human MAC in the upper chamber in normal (NG) or high (HG) glucose concentration. After cross‐talk, the conditioned media and cells were isolated and investigated for the expression of MMPs, MCP‐1 and signalling molecules. We found that upon cross‐talk with MAC in HG, SMC exhibit: (i) augmented expression of MMP‐1 and MMP‐9; (ii) significant increase in the enzymatic activity of MMP‐9; (iii) higher levels of soluble MCP‐1 chemokine which is functionally active and involved in MMPs up‐regulation; (iv) activated PKCα signalling pathway which, together with NF‐kB are responsible for MMP‐1 and MMP‐9 up‐regulation, and (v) impaired function of collagen assembly. Taken together, our data indicate that MCP‐1 released by cell cross‐talk in diabetic conditions binds to CCR2 and triggers MMP‐1 and MMP‐9 over‐expression and activity, features that could explain the high vulnerability of atherosclerotic plaque found at diabetic patients.     

Insulin induces the low density lipoprotein receptor‐related protein 1 (LRP1) degradation by the proteasomal system in J774 macrophage‐derived cells

Low‐density lipoprotein receptor‐related protein 1 (LRP1) is an endocytic receptor, which binds and internalizes diverse ligands such as activated α₂‐macroglobulin (α₂M*). LRP1 promotes intracellular signaling, which downstream mediates cellular proliferation and migration of different types of cells, including macrophages. Unlike the LDL receptor, LRP1 expression is not sensitive to cellular cholesterol levels but appears to be responsive to insulin. It has been previously demonstrated that insulin increases the cell surface presentation of LRP1 in adipocytes and hepatocytes, which is mediated by the intracellular PI₃K/Akt signaling activation. The LRP1 protein distribution is similar to other insulin‐regulated cell surface proteins, including transferring receptor (Tfr). However, in macrophages, the insulin effect on the LRP1 distribution and expression is not well characterized. Considering that macrophages play a central role in the pathogenesis of atherosclerosis, herein we evaluate the effect of insulin on the cellular expression of LRP1 in J774 macrophages‐derived cells using Western blot and immunofluorescence microscopy. Our data demonstrate that insulin induces a significant decrease in the LRP1 protein content, without changing the specific mRNA level of this receptor. Moreover, insulin specifically affected the protein expression of LRP1 but not Tfr. The insulin‐induced protein degradation of LRP1 in J774 cells was mediated by the activation of the PI₃K/Akt pathway and proteasomal system by an enhanced ubiquitin–receptor conjugation. The decreased content of LRP1 induced by insulin affected the cellular internalization of α₂M*. Thus, we propose that the protein degradation of LRP‐1 induced by insulin in macrophages could have important effects on the pathogenesis of atherosclerosis. J. Cell. Biochem. 106: 372–380, 2009. © 2008 Wiley‐Liss, Inc.     

Haplotype analyses, mechanism and evolution of common double mutants in the human LDL receptor gene

Familial hypercholesterolemia (FH), an autosomal dominant inherited disorder resulting in increased levels of circulating plasma low-density lipoprotein (LDL), tendon xanthomas and premature coronary artery disease (CAD), is caused by defects in the LDL receptor gene (LDLR). Three widespread LDLR alterations not causing FH (c.1061-8T>C, c.2177C>T and c.829G>A) and one mutation (c.12G>A) with narrow geographical distribution and thought to cause disease were investigated. In an attempt to improve knowledge on their origin, spread and possible selective effects, estimations of the ages of these variants (t generations) and haplotype analysis were performed by genotyping 86 healthy individuals and 98 FH patients in Spain for five LDLR SNPs: c.81T>C, c.1413G>A, c.1725C>T, c.1959T>C, and c.2232G>A; most patients carried two of these LDLR variants simultaneously. It was found that both the c.1061-8T>C (t = 54) and c.2177C>T alterations (t = 62) arose at about the same time (54 and 62 generations ago, respectively) in the CGCTG haplotype, while the c.12G>A mutation (t = 70) appeared in a CGCCG haplotype carrying an earlier c.829G>A alteration (t = 83). The estimated ages of selectively neutral alterations could explain their distribution by migrations. The origin of the c.12G>A mutation could be in the Iberian Peninsula; despite its estimated age, a low selective pressure could explain its conservation in Spain from where it could have spread to China and Mexico, since the sixteenth century through the Spanish/Portuguese colonial expeditions.     

Effect of irreversibly glycated LDL in human vascular smooth muscle cells: lipid loading,oxidative and inflammatory stress

The major complication of diabetes is accelerated atherosclerosis, the progression of which entails complex interactions between the modified low‐density lipoproteins (LDL) and the cells of the arterial wall. Advanced glycation end product‐modified‐LDL (AGE‐LDL) that occurs at high rate in diabetes contributes to diabetic atherosclerosis, but the underlying mechanisms are not fully understood. The aim of this study was to assess the direct effect of AGE‐LDL on human vascular smooth muscle cells (hSMC) dysfunction. Cultured hSMC incubated (24 hrs) with human AGE‐LDL, native LDL (nLDL) or oxidized LDL (oxLDL) were subjected to: (i) quantification of the expression of the receptors for modified LDL and AGE proteins (LRP1, CD36, RAGE) and estimation of lipid loading, (ii) determination of NADPH oxidase activity and reactive oxygen species (ROS) production and (iii) evaluation of the expression of monocyte chemoattractant protein‐1 (MCP‐1). The results show that exposure of hSMC to AGE‐LDL (compared to nLDL) induced: (a) increased NADPH oxidase activity (30%) and ROS production (28%) by up‐regulation of NOX1, NOX4, p22phox and p67phox expression, (b) accumulation of intracellular cholesteryl esters, (c) enhanced gene expression of LRP1 (160%) and CD36 (35%), and protein expression of LRP1, CD36 and RAGE, (d) increased MCP‐1 gene expression (160%) and protein secretion (300%) and (e) augmented cell proliferation (30%). In conclusion, AGE‐LDL activates hSMC (increasing CD36, LRP1, RAGE), inducing a pro‐oxidant state (activation of NADPHox), lipid accumulation and a pro‐inflammatory state (expression of MCP‐1). These results may partly explain the contribution of AGE‐LDL and hSMC to the accelerated atherosclerosis in diabetes.     

Cascade screening and genetic diagnosis of familial hypercholesterolemia in clusters of the Southeastern region from Brazil

Familial hypercholesterolemia (FH) is an autosomal dominant genetic disease characterized by high levels of low-density lipoprotein-cholesterol (LDLc), associated to premature cardiovascular disease. The detection of the variants related to FH is important to improve the early diagnosis in probands / index-cases (ICs) and their relatives. We included ICs with FH and their relatives, living in a small region of Minas Gerais state-Brazil, which were classified according to Dutch Lipid Clinic Network Criteria (DLCNC) and submitted to sequencing of genes related to FH (LDLR, APOB, PCSK9, LDLRAP1, LIPA, STAP1, APOE, ABCG5 e ABCG8). In a total of 143 subjects (32 ICs and 111 relatives), eight variants were identified in 91 individuals. From these variants, five were in LDLR [p.(Asp224Asn), p.(Ser854Gly), p.(Cys34Arg), p.(Asp601His), deletion of exon15 in LDLR)], one in APOB [p.(Met499Val)], one in PCSK9 [p.(Arg237Trp)] and one in APOE [p.(Pro28Leu)] genes. The variants were detected in 100% of those subjects classified as definitive, 87% as probable and 69% as possible FH cases based on DLCNC. The LDLc level was higher in individuals with corneal arch and xanthomas or xanthelasmas, as well as in pathogenic or probably pathogenic variants carriers. This study showed higher frequency of LDLR gene variants compared to other genes related to LDL metabolism in individuals with FH in Minas Gerais – Brazil and the presence of FH in relatives without previous diagnosis. Our data reinforce the importance of molecular and clinical evaluation of FH relatives in order to early diagnosis the FH, as well as cardiovascular diseases prevention.

     

Role of the low‐density lipoprotein receptor‐related protein‐1 in regulation of chondrocyte differentiation

The low‐density lipoprotein receptor‐related protein 1 (LRP1) is known as an endocytic and signal transmission receptor. We formerly reported the gene expression and the localization of LRP1 in cartilage tissue and chondrocytes, but its roles in the differentiation of chondrocytes remained to be investigated. Here, in order to address this issue, we employed RNAi strategy to knockdown lrp1 in chondrocytic cells and obtained findings indicating a critical role therein. As a result of lrp1 knockdown, aggrecan and col2a1 mRNA levels were decreased. However, that of col10a1 or mmp13 mRNA was rather increased. Under this condition, we performed a promoter assay for Axin2, which is known to be induced by activation of the WNT/β‐catenin (βcat) signaling pathway. Thereby, we found that Axin2 promoter activity was enhanced in the lrp1 knockdown cells. Furthermore, when the WNT/β–catenin pathway was activated in chondrocytic cells by WNT3a or SB216763, which inhibits the phosphorylation of GSK3β, the mRNA levels of aggrecan and col2a1 were decreased, whereas that of mmp13 was increased. Additionally, the level of phosphorylated protein kinase C (PKC) ζ was also decreased in the lrp1 knockdown cells. When the phosphorylation of PKCζ was selectively inhibited, aggrecan and col2a1 mRNA levels decreased, whereas the mmp13 mRNA level increased. These data demonstrate that LRP1 exerts remarkable effects to retain the mature phenotype of chondrocytes as a critical mediator of cell signaling. Our findings also indicate that the onset of hypertrophy during endochondral ossification appears to be particularly dependent on the WNT and PKC signaling initiated by LRP1. J. Cell. Physiol. 222:138–148, 2010. © 2009 Wiley‐Liss, Inc.     

Protective effects of green tea polyphenols and their major component, (–)-epigallocatechin-3-gallate (EGCG), on 6-hydroxydopamine-induced apoptosis in PC12 cells

SUMMARY

Oxidative modification of low density lipoprotein (LDL) appears to be important in the pathogenesis of atherosclerosis. Inhibiting the oxidation of LDL may retard or prevent the atherogenic process. However, susceptibility of LDL to oxidation in vitro and its atherogenicity in vivo may not always correlate. Subjects with familial hypercholesterolaemia (FH) develop severe, premature atherosclerosis despite having large, bouyant LDL particles which are less susceptible to oxidation. High dose, long-term vitamin E increases the resistance of LDL to oxidation but, unlike probucol, has no effect on xanthoma regression in homozygous FH. In FH, the quantity of LDL takes priority and the main aim of therapy is reduction of LDL bulk. Individuals with small, dense LDL particles are at increased risk for atherosclerosis despite desirable plasma LDL cholesterol levels. Small, dense LDL particles are more susceptible to oxidation and in these subjects antioxidant therapy may be of greater benefit. In subjects with atherosclerosis, current management should be aimed primarily at reducing the LDL cholesterol level. In the future antioxidant therapy may complement our management of hypercholesterolaemia.     

Blood treatment of Lyme borreliae demonstrates the mechanism of CspZ‐mediated complement evasion to promote systemic infection in vertebrate hosts

Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most common vector‐borne disease in the United States and Europe. The spirochetes are transmitted from mammalian and avian reservoir hosts to humans via ticks. Following tick bites, spirochetes colonize the host skin and then disseminate haematogenously to various organs, a process that requires this pathogen to evade host complement, an innate immune defence system. CspZ, a spirochete surface protein, facilitates resistance to complement‐mediated killing in vitro by binding to the complement regulator, factor H (FH). Low expression levels of CspZ in spirochetes cultivated in vitro or during initiation of infection in vivo have been a major hurdle in delineating the role of this protein in pathogenesis. Here, we show that treatment of B. burgdorferi with human blood induces CspZ production and enhances resistance to complement. By contrast, a cspZ‐deficient mutant and a strain that expressed an FH‐nonbinding CspZ variant were impaired in their ability to cause bacteraemia and colonize tissues of mice or quail; virulence of these mutants was however restored in complement C3‐deficient mice. These novel findings suggest that FH binding to CspZ facilitates B. burgdorferi complement evasion in vivo and promotes systemic infection in vertebrate hosts.     

Tight junction proteins expression and modulation in immune cells and multiple sclerosis

The tight junction proteins (TJPs) are major determinants of endothelial cells comprising physiological vascular barriers such as the blood–brain barrier, but little is known about their expression and role in immune cells. In this study we assessed TJP expression in human leukocyte subsets, their induction by immune activation and modulation associated with autoimmune disease states and therapies. A consistent expression of TJP complexes was detected in peripheral blood leukocytes (PBLs), predominantly in B and T lymphocytes and monocytes, whereas the in vitro application of various immune cell activators led to an increase of claudin 1 levels, yet not of claudin 5. Claudins 1 and 5 levels were elevated in PBLs of multiple sclerosis (MS) patients in relapse, relative to patients in remission, healthy controls and patients with other neurological disorders. Interestingly, claudin 1 protein levels were elevated also in PBLs of patients with type 1 diabetes (T1D). Following glucocorticoid treatment of MS patients in relapse, RNA levels of JAM3 and CLDN5 and claudin 5 protein levels in PBLs decreased. Furthermore, a correlation between CLDN5 pre‐treatment levels and clinical response phenotype to interferon‐β therapy was detected. Our findings indicate that higher levels of leukocyte claudins are associated with immune activation and specifically, increased levels of claudin 5 are associated with MS disease activity. This study highlights a potential role of leukocyte TJPs in physiological states, and autoimmunity and suggests they should be further evaluated as biomarkers for aberrant immune activity and response to therapy in immune‐mediated diseases such as MS.     

Fasting Plasma Insulin Modulates Lipid Levels and Particle Sizes in 2‐ to 3‐Year‐Old Children

Objective: Obesity and hyperinsulinemia are associated with dyslipidemia in adults and older children, but little is known about these relationships in very young children. We examined the relation of fasting insulin to lipid levels and lipid particle size in young healthy children. Research Methods and Procedures: Analyses were performed on data from 491 healthy 2‐ and 3‐year old Hispanic children enrolled in a dietary study conducted in New York City, 1992–1995. Obesity measures included BMI, ponderal index, skinfold thickness, and waist circumference. Low‐density lipoprotein (LDL)‐ and high‐density lipoprotein (HDL)‐cholesterol particle size were measured by nuclear magnetic resonance. Results: Fasting insulin level was positively correlated with triglyceride levels (r = 0.24 for boys and r = 0.23 for girls; p < 0.001 for both) and inversely correlated with HDL‐cholesterol level in boys (r = ?0.20; p < 0.01). Higher fasting insulin level was also correlated with smaller mean HDL particle size in both boys (r = ?0.21; p < 0.001) and girls (r = ?0.14; p < 0.05) and smaller mean LDL particle size in boys (r = ?0.13; p < 0.05). The associations of fasting insulin level with triglyceride and HDL‐cholesterol levels and HDL and LDL particle size remained significant after multivariate regression adjustment for age, sex, and BMI or ponderal index. Discussion: Fasting insulin level is associated with relative dyslipidemia in healthy 2‐ and 3‐year‐old Hispanic children.     

Relationship among LRP1 expression,Pyk2 phosphorylation and MMP‐9 activation in left ventricular remodelling after myocardial infarction

Left ventricular (LV) remodelling after myocardial infarction (MI) is a crucial determinant of the clinical course of heart failure. Matrix metalloproteinase (MMP) activation is strongly associated with LV remodelling after MI. Elucidation of plasma membrane receptors related to the activation of specific MMPs is fundamental for treating adverse cardiac remodelling after MI. The aim of current investigation was to explore the potential association between the low‐density lipoprotein receptor‐related protein 1 (LRP1) and MMP‐9 and MMP‐2 spatiotemporal expression after MI. Real‐time PCR and Western blot analyses showed that LRP1 mRNA and protein expression levels, respectively, were significantly increased in peri‐infarct and infarct zones at 10 and 21 days after MI. Confocal microscopy demonstrated high colocalization between LRP1 and the fibroblast marker vimentin, indicating that LRP1 is mostly expressed by cardiac fibroblasts in peri‐infarct and infarct areas. LRP1 also colocalized with proline‐rich tyrosine kinase 2 (pPyk2) and MMP‐9 in cardiac fibroblasts in ischaemic areas at 10 and 21 days after MI. Cell culture experiments revealed that hypoxia increases LRP1, pPyk2 protein levels and MMP‐9 activity in fibroblasts, without significant changes in MMP‐2 activity. MMP‐9 activation by hypoxia requires LRP1 and Pyk2 phosphorylation in fibroblasts. Collectively, our in vivo and in vitro data support a major role of cardiac fibroblast LRP1 levels on MMP‐9 up‐regulation associated with ventricular remodelling after MI.     

Identification of a recurrent insertion mutation in the LDLR gene in a Pakistani family with autosomal dominant hypercholesterolemia

Familial Hypercholesterolemia (FH) results in elevated levels of blood lipids including total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) with normal triglycerides (TG). This disease is one of the major contributors towards an early onset of coronary heart disease (CHD). The aim of the present study was to identify the genes responsible for causing FH in Pakistani population, for this purpose a large consanguineous FH family was selected for genetic analysis. Serum lipid levels, including TC, TG, LDL-C and high density lipoprotein cholesterol (HDL-C), were determined in patients and healthy controls. In order to find the causative mutation in this family, direct sequencing of the low density lipoprotein receptor (LDLR) gene was performed. In addition the part of the Apolipoprotein-B (APOB) gene containing the mutations R3500Q and R3500W was also sequenced. Affected individuals of the family were found to have raised TC and LDL-C levels. Sequencing revealed an insertion mutation (c.2416_2417InsG) in exon 17 of the LDLR gene in all the affected individuals of the family. Common FH causing APOB mutations were not present in this family. Heterozygous individuals had TC levels ranging from ~300–500 mg/dl and the only homozygous individual with typical xanthomas had TC levels exceeding 900 mg/dl. This is the first report of a known LDLR gene mutation causing FH in the Pakistani population. Despite a large heterogeneity of LDLR mutations there are still some common mutations which are responsible for FH throughout the world.     

The laying hen expresses two different low density lipoprotein receptor-related proteins

We have identified, by a combination of ligand, 45Ca2+, and immunoblotting, two large membrane proteins akin to the mammalian so-called low density lipoprotein (LDL) receptor-related protein (LRP) in chicken tissues. LRP has thus far been demonstrated only in mammalian species where it is thought to act as a receptor for proteinase-alpha 2-macroglobulin complexes and/or chylomicron remnants, lipoproteins not produced in birds. One of the chicken LRPs was demonstrated in liver, and has the same apparent Mr and hallmark biochemical properties as rat liver LRP. The other chicken LRP is smaller (approximately 380 kDa) and is expressed in ovarian follicles, but is undetectable in liver. Immunological analysis demonstrated a lack of cross-reactivity between the two LRPs, as well as between them and the previously identified chicken oocyte-specific 95-kDa receptor for the yolk precursors, very low density lipoprotein, and vitellogenin (Stifani, S., Barber, D. L., Nimpf, J., and Schneider, W. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 87, 1955-1959). As shown by ligand blotting, both chicken LRPs have the ability to interact with vitellogenin, a property they share not only with rat LRP, but also with mammalian LDL receptors. To obtain independent confirmation of the ligand blotting results, the smaller (follicular) LRP was purified and high-affinity binding of vitellogenin to it was demonstrated by a solid-phase filtration binding assay. Amino acid sequences of tryptic fragments of the smaller LRP were obtained, and its homology with human LRP demonstrated through unambiguous alignment of three fragments. Both chicken LRPs, the chicken oocyte 95-kDa receptor, as well as rat LRP, could be shown by ligand blotting to interact specifically with chicken serum alpha 2-macroglobulin. In addition, human apolipoprotein E, a ligand implicated in receptor-mediated metabolism of chylomicron remnants, also binds to the smaller chicken LRP, further emphasizing the similarities between LDL receptors and related proteins from a variety of species. In analogy to the known dichotomy of chicken LDL receptors, which is characterized by the production of the 95-kDa oocyte-specific receptor on one hand and a 130-kDa LDL receptor that is exclusively expressed in somatic cells (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139), it appears that the smaller and larger chicken LRPs also may be restricted to the oocyte and somatic cells, respectively.     

Expression of Recombinant LDLR–EGFP Fusion Protein in HEK-293 Cells as a Promising Tool to Assess the Effect of <Emphasis Type="Italic">LDLR</Emphasis> Gene Mutations

Mutations in the low density lipoprotein receptor gene (LDLR) frequently impair folding and intracellular traffic of the receptor protein, resulting in the development of a monogenic disorder, familial hypercholesterolemia (FH). Identification of novel LDLR mutations requires confirmation of their functional importance in distinguishing pathogenic mutations from neutral changes in the aminoacid sequence. To elaborate a system for evaluation of the effect of mutation on the folding and intracellular transport of the LDLR, as well as its ability to bind low density lipoprotein (LDL), we constructed a plasmid containing LDLR cDNA and the gene of enhanced green fluorescent protein (EGFP). Confocal microscopy has shown that, upon transient transfection of HEK293 cells with the plasmid, the recombinant fusion protein LDLR–EGFP is transported onto the cellular membrane and binds labeled LDL. This construct will be further modified by site-directed mutagenesis to reproduce the LDLR missense mutations most common in the population of northwest Russia so as to study the subcellular localization and function of the modified chimeric protein.     

Primary hypercholesterolaemia impairs glucose homeostasis and insulin secretion in low-density lipoprotein receptor knockout mice independently of high-fat diet and obesity

We investigated whether primary hypercholesterolaemia per se affects glucose homeostasis and insulin secretion in low-density lipoprotein receptor knockout mice (LDLR^−/−). Glucose plasma levels were increased and insulin decreased in LDLR^−/− compared to the wild-type mice. LDLR^−/− mice presented impaired glucose tolerance, but normal whole body insulin sensitivity. The dose–response curve of glucose-stimulated insulin secretion was shifted to the right in LDLR^−/− islets. Significant reductions in insulin secretion in response to l-leucine or 2-ketoisocaproic acid were also observed in LDLR^−/−. Islet morphometric parameters, total insulin and DNA content were similar in both groups. Glucose uptake and oxidation were reduced in LDLR^−/− islets. Removal of cholesterol from LDLR^−/− islets corrected glucose-stimulated insulin secretion. These results indicate that enhanced membrane cholesterol content due to hypercholesterolaemia leads to a lower insulin secretion and glucose intolerance without affecting body insulin sensitivity. This represents an additional risk factor for diabetes and atherosclerosis in primary hypercholesterolaemia.     

Measures of immune system status in young‐of‐the‐year winter flounder Pseudopleuronectes americanus

The immune status of young‐of‐the‐year (YOY) winter flounder Pseudopleuronectes americanus was evaluated in fish collected from six areas around Long Island, NY, U.S.A. representing more urban areas with high population density in the west, to less densely populated more rural areas in to the east. Gene expression markers for innate immunity (pleurocidin) and contaminant exposure (cytochrome P4501A; cyp1a) were measured in liver and fin of fish collected at each site. Expression of pleurocidin was significantly higher in fin than liver, but was highly variable among individuals. Some statistically significant differences in pleurocidin expression among sites were observed, although elevated levels were not associated with degree of urbanization. Expression was related in part to fish size: a positive correlation between expression and total length (L_T) of fish was observed with the largest L_T class (>125 mm) exhibiting significantly elevated pleurocidin expression as compared with fish in the smaller L_T class. This indicates that immune competency may increase with age. No site‐specific differences in cyp1a expression were observed. These data suggest that exposure to aromatic hydrocarbon contaminants is fairly widespread throughout the study area and that any differences in pleurocidin expression in YOY P. americanus are probably due to other factors. Antimicrobial activity was also measured as a functional indicator of immune response. Activity was highly variable, showing no significant site‐specific differences, and no significant correlation to pleurocidin expression. The lack of correlation between pleurocidin expression and antimicrobial activity indicates that other antimicrobial peptides may be active against the bacteria tested or that other factors are influencing antimicrobial activity. This is the first quantitative evaluation of pleurocidin expression in YOY P. americanus from an urban area. Further work is needed to characterize factors controlling pleurocidin expression, as well as other indicators of immune response in young fish.