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Very-low-density lipoprotein receptor deficient mice are protected against diet-induced obesity and insulin resistance
Thursday June 29, 2000: Workshop Abstracts W:34 Lipoprotein Receptors I ThW3:34 ] Roles of ApoE receptors and their family Tokuo Yamamoto. Tohoku University Gene Research Center, Sendal Japan ApoE plays a key role in the transportation and metabolism of plasma cholesterol and triacylglyeerol. It is recognized by several receptors including the LDL receptor, LDL receptor related proteins, VLDL receptor (VLDLR), and apoE receptor 2 (apoER2). VLDLR and apoER2 consist of five common functional domains that resemble LDLR. Although they are structurally closely related, their expressions are completely different: VLDLR mRNA is abundant in heart and skeletal muscle, and apoER2 mRNA predominates in brain, but they are almost absent in the liver. In chicken, VLDLR is expressed almost exclusively in oocytes and mediates the uptake of yolk precursors, VLDL and vitellogenin. Chicken mutants lacking VLDLR are sterile and exhibit severe hyperlipidemia, demonstrating that VLDLR is crucial in non-mammalian vertebrate oogenesis. In contrast to nonmarnmalian vertebrates, mice lacking VLDLR exhibit modest decreases in body weight, body mass index and adipose tissue mass, while their plasma cholesterol levels, triacylglycerol levels, and lipoprotein profiles are not altered. Furthermore, knockout mice lacking both VLDLR and LDLR exhibit a modest hypercholesterolemia, whereas apoE knockout mice exhibit a profound hypercholesterolemia. These data suggests the presence of several apoE receptors. To identify these, we characterized several cDNAs containing the ligand-binding region. Our recent characterization on apoE receptors and their family proteins will be presented.
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ntracellular trafficking - Consequence of receptor mediated uptake of lipoproteins
U. Beisiegel, J. Heeren. Medical Clinic, UKE, Hamburg, Germany Objective: To study the intracellular pathway of chylomicron remnant (CR) constituents after endocytosis by lipoprotein receptors, most probably by the LDL receptor-related protein (LRP). Methods: Human chylomicrons were isolated from Apo C-II deficient patients and hydrolyzed in vitro by lipoprotein lipase (LpL) to obtain CR. The CR were labeled with iodine and used for biochemical studies (binding, uptake, degradation and recycling) in human hepatoma cells and human fibroblasts with and without LDL receptor (LDLR). In addition native CR and CR labeled with Dil (fluorescent phospholipid analogue) were incubated on the same cells for immunofluorescence analysis. Results: The biochemical studies showed that apo E and apo C were not degraded, in contrast to LDL-apo B. The labeled apoproteins remained in the cell and apo E was detected within peripheral endosomal compamnents. In the presence of HDL, as an extracellular acceptor, the radioactivity was re-secreted into the medium and labeled apo E was associated with HDL. The Dil-label, in contrast, was found in the lysosomal compartment. This intracellular pathway which leads to lipoprotein disassembly occurred in both LDLR positive and negative cells, suggesting that LRP is a candidate receptor for CR recycling. Condusions: The uptake of human CR, possibly via LRP, leads to the intracellular disassembly of the particles. The surface constituents, mainly apo E, are recycled when HDL is present as an extracelhilar acceptur.
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TGF-fll increases the expression of lectine-like oxidized LDL reeeptor-1 (LOX-1)
M. Minami, N. Kume, H. Kataoka, M. Morimoto, T. IOta. Department of Geriatric Medicine, Kyoto University, Kyoto, Japan
Objective: LOX-I is a novel membrane receptor for atherogenic oxidized LDL, which can be expressed by vascular endothelial cells and macrophages. We have examined whether TGF-fll, which inhibits the expression of class A scavenger receptors in macrophages and plays crucial roles in vascular diseases including atherosclerosis, can induce expression of LOX-1. Methods: 1. Cultured bovine aortic endothelial (BAEC) or smooth muscle (BASMC) cells were treated with recombinant human TGF-flL (0.1-10 ng/ml), then immunoblot analyses and Northern blot analyses were performed. 2. After pretreatment with or without actinomycin D (2.5-5/zg/ml) for 30 minutes, BAEC or BASMC were treated with 1 ng/ml of TGF-flb then Northern blot analyses were performed. 3. Murine peritoneal macrophages were cultured overnight and treated with TGF-~l (0.1-10 ng/ml), and then Northern blot analyses were performed. Results: L Immunoblot and Northern blot analyses showed that treatment with TGF-fll increased the expression of LOX-1 protein and mRNA in both BAEC and BASMC. Treatment with 1 ng/ml of TGF-fll for 8 hours resulted in 4.2-fold and 2.g-fold increases in the amounts of LOX-1 protein
241
in BAEC and BASMC, respectively. 2. Pretreatment with actinomycin D completely abolished LOX-1 mRNA induction elicited by TGF-31, suggesting that TGF-fll may stimulate transcription of the LOX-1 gene. 3. TGF-/~l also induced LOX-1 expression in mudne peritoneal macrophages in a doseand time-dependent fashion; treatment with 10 ng/ml of TGF-~61 for 4 hours resulted in a 4.7-fold increase in the amount of LOX-1 mRNA. Conclusions: TGF-/~I can highly induce LOX-1 expression in vascular endothelial cells, smooth muscle cells and macrophages. TGF-/~I appears one of the key regulators that can modulate the expression of scavenger receptors. I ThW6:34 [ Very-low-density lipoprotein receptor deficient mice are
protected against diet-induced obesity and insulin resistance J.R. Goudriaan I , M.C. Jong ] , V.E.H. Dahlmans 1, P.J. Tacken2, L.M. Havekes I . 1Gaubius Lab; 2Leiden Univ Med Ctr, Leiden, Netherlands Objective: To investigate whether very-low-density lipoprotein receptor deficient (VLDLR - / - ) mice develop a clear phenotype when fed a high-fat diet. Methods: Homozygous knockout male mice that lack the VLDL receptor and wild-type littermates were fed a high-fat diet (I-IF; 46% of the calories are provided by corn oil) for a period of 17 weeks. Their body weight, food intake, plasma insulin, lipid, and ketone levels, glucose tolerance, intestinal fat absorption and the fatty acid composition of liver, heart, muscle and fat tissues were measured. Results: After 17 weeks on the HF diet, VLDLR - / - mice showed a slight increase in their body weight (from 25.3 -4- 2.3 g to 29.7 4- 4.8 g), whereas wild-type littermates became obese (from 28.7 4- 1.6 g to 44.1 4- 5.7 g). No differences were observed with respect to food intake. Furthermore after 17 weeks of HF feeding, VLDLR - / - mice exhibited significant lower plasma insulin levels as compared to wild-type mice (1.3 4- 0.8 versus 4.6 + 2.5 ng/ml) and were able to clear an intraperitoneal injected glucose bolus more rapidly than wild-type mice. These results clearly indicate that VLDLR - / mice are protected against HF diet-induced insulin resistance. There were no significant differences found between VLDLR - / - mice and their controls with respect to plasma lipids, fatty acid oxidation (ketone bodies), intestinal fat absorption or fatty acid tissue-composition. Conclusion: In contrast to their wild-type littermates, VLDLR - / - mice do not become obese, nor insulin resistant after 17 weeks of high-fat feeding. These findings are indicative for a fundamental role of the VLDL receptor in the entry of fat-derived calories into tissues.
I ThW7:34 I Evidence for the role of megalin in rensI resorption F
of
transthyretin
M.M. Sousa 1,2, A.G. Norden3, C. Jacobsen4, P.J. Verroust5, S.K. Moestrup4 , M.J. Saraiva 1'2 . l Amyloid Unit, Instituto de Biologia Molecular e Celular; 21nstituto de Ci~ncias Biomddicas Abel Salazar, University of Porto, Portugal; 3Department of Chemical Pathology, Chase Farm Hospitals Trust, Middlesex, UK; 4 Department of Medical Biochemistry, University of Aarrhus, Denmark; ~lnstitut National de la Santd et de la Recherche Medicale, U489, Hopital Tenon, Paris, France Megalin is expressed on the luminal surface of various epithelia including the renal proximal tubules. In the kidney it plays an important role in tubular uptake of macromolecules filtered through the glomerulus. By two-dimensional gel electrophoresis followed by mass spectrometry analysis we identified transthyretin (TTR) as an abundant protein in the urine of patients with renal tubule failure. TTR is a plasma protein that functions as a transporter of thyroxine and retinol. It was recently shown that by binding RBP, megalin has an essential role in transepithelial transport of retinol. It is possible that in vivo, megalin might also be responsible for the tubular resorption of TTR. To investigate this hypothesis we performed different TTR binding assays by surface plasmon ressonance (SPR) analysis and by using immortalized rat yolk sac cells with high expression levels of megalin. Binding of purified TTR, free as well as in complex with thyroxine or retinol, to immobilized megalin was shown by the SPR analysis. Radiolabeled TTR was rapidly taken up by cultured cells and the uptake was partially inhibited by a polyclonal megalin antibody and by the receptor-associated protein (RAP), a chaperone-like protein inhibiting ligand binding to megalin and other LDLr family receptors. The present data indicate that T'I'R represents a novel megalin ligand of potential importance in the transepithelial transport of retinol and thyroxine.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
~
ntracellular trafficking - Consequence of receptor mediated uptake of lipoproteins
U. Beisiegel, J. Heeren. Medical Clinic, UKE, Hamburg, Germany Objective: To study the intracellular pathway of chylomicron remnant (CR) constituents after endocytosis by lipoprotein receptors, most probably by the LDL receptor-related protein (LRP). Methods: Human chylomicrons were isolated from Apo C-II deficient patients and hydrolyzed in vitro by lipoprotein lipase (LpL) to obtain CR. The CR were labeled with iodine and used for biochemical studies (binding, uptake, degradation and recycling) in human hepatoma cells and human fibroblasts with and without LDL receptor (LDLR). In addition native CR and CR labeled with Dil (fluorescent phospholipid analogue) were incubated on the same cells for immunofluorescence analysis. Results: The biochemical studies showed that apo E and apo C were not degraded, in contrast to LDL-apo B. The labeled apoproteins remained in the cell and apo E was detected within peripheral endosomal compamnents. In the presence of HDL, as an extracellular acceptor, the radioactivity was re-secreted into the medium and labeled apo E was associated with HDL. The Dil-label, in contrast, was found in the lysosomal compartment. This intracellular pathway which leads to lipoprotein disassembly occurred in both LDLR positive and negative cells, suggesting that LRP is a candidate receptor for CR recycling. Condusions: The uptake of human CR, possibly via LRP, leads to the intracellular disassembly of the particles. The surface constituents, mainly apo E, are recycled when HDL is present as an extracelhilar acceptur.
•
TGF-fll increases the expression of lectine-like oxidized LDL reeeptor-1 (LOX-1)
M. Minami, N. Kume, H. Kataoka, M. Morimoto, T. IOta. Department of Geriatric Medicine, Kyoto University, Kyoto, Japan
Objective: LOX-I is a novel membrane receptor for atherogenic oxidized LDL, which can be expressed by vascular endothelial cells and macrophages. We have examined whether TGF-fll, which inhibits the expression of class A scavenger receptors in macrophages and plays crucial roles in vascular diseases including atherosclerosis, can induce expression of LOX-1. Methods: 1. Cultured bovine aortic endothelial (BAEC) or smooth muscle (BASMC) cells were treated with recombinant human TGF-flL (0.1-10 ng/ml), then immunoblot analyses and Northern blot analyses were performed. 2. After pretreatment with or without actinomycin D (2.5-5/zg/ml) for 30 minutes, BAEC or BASMC were treated with 1 ng/ml of TGF-flb then Northern blot analyses were performed. 3. Murine peritoneal macrophages were cultured overnight and treated with TGF-~l (0.1-10 ng/ml), and then Northern blot analyses were performed. Results: L Immunoblot and Northern blot analyses showed that treatment with TGF-fll increased the expression of LOX-1 protein and mRNA in both BAEC and BASMC. Treatment with 1 ng/ml of TGF-fll for 8 hours resulted in 4.2-fold and 2.g-fold increases in the amounts of LOX-1 protein
241
in BAEC and BASMC, respectively. 2. Pretreatment with actinomycin D completely abolished LOX-1 mRNA induction elicited by TGF-31, suggesting that TGF-fll may stimulate transcription of the LOX-1 gene. 3. TGF-/~l also induced LOX-1 expression in mudne peritoneal macrophages in a doseand time-dependent fashion; treatment with 10 ng/ml of TGF-~61 for 4 hours resulted in a 4.7-fold increase in the amount of LOX-1 mRNA. Conclusions: TGF-/~I can highly induce LOX-1 expression in vascular endothelial cells, smooth muscle cells and macrophages. TGF-/~I appears one of the key regulators that can modulate the expression of scavenger receptors. I ThW6:34 [ Very-low-density lipoprotein receptor deficient mice are
protected against diet-induced obesity and insulin resistance J.R. Goudriaan I , M.C. Jong ] , V.E.H. Dahlmans 1, P.J. Tacken2, L.M. Havekes I . 1Gaubius Lab; 2Leiden Univ Med Ctr, Leiden, Netherlands Objective: To investigate whether very-low-density lipoprotein receptor deficient (VLDLR - / - ) mice develop a clear phenotype when fed a high-fat diet. Methods: Homozygous knockout male mice that lack the VLDL receptor and wild-type littermates were fed a high-fat diet (I-IF; 46% of the calories are provided by corn oil) for a period of 17 weeks. Their body weight, food intake, plasma insulin, lipid, and ketone levels, glucose tolerance, intestinal fat absorption and the fatty acid composition of liver, heart, muscle and fat tissues were measured. Results: After 17 weeks on the HF diet, VLDLR - / - mice showed a slight increase in their body weight (from 25.3 -4- 2.3 g to 29.7 4- 4.8 g), whereas wild-type littermates became obese (from 28.7 4- 1.6 g to 44.1 4- 5.7 g). No differences were observed with respect to food intake. Furthermore after 17 weeks of HF feeding, VLDLR - / - mice exhibited significant lower plasma insulin levels as compared to wild-type mice (1.3 4- 0.8 versus 4.6 + 2.5 ng/ml) and were able to clear an intraperitoneal injected glucose bolus more rapidly than wild-type mice. These results clearly indicate that VLDLR - / mice are protected against HF diet-induced insulin resistance. There were no significant differences found between VLDLR - / - mice and their controls with respect to plasma lipids, fatty acid oxidation (ketone bodies), intestinal fat absorption or fatty acid tissue-composition. Conclusion: In contrast to their wild-type littermates, VLDLR - / - mice do not become obese, nor insulin resistant after 17 weeks of high-fat feeding. These findings are indicative for a fundamental role of the VLDL receptor in the entry of fat-derived calories into tissues.
I ThW7:34 I Evidence for the role of megalin in rensI resorption F
of
transthyretin
M.M. Sousa 1,2, A.G. Norden3, C. Jacobsen4, P.J. Verroust5, S.K. Moestrup4 , M.J. Saraiva 1'2 . l Amyloid Unit, Instituto de Biologia Molecular e Celular; 21nstituto de Ci~ncias Biomddicas Abel Salazar, University of Porto, Portugal; 3Department of Chemical Pathology, Chase Farm Hospitals Trust, Middlesex, UK; 4 Department of Medical Biochemistry, University of Aarrhus, Denmark; ~lnstitut National de la Santd et de la Recherche Medicale, U489, Hopital Tenon, Paris, France Megalin is expressed on the luminal surface of various epithelia including the renal proximal tubules. In the kidney it plays an important role in tubular uptake of macromolecules filtered through the glomerulus. By two-dimensional gel electrophoresis followed by mass spectrometry analysis we identified transthyretin (TTR) as an abundant protein in the urine of patients with renal tubule failure. TTR is a plasma protein that functions as a transporter of thyroxine and retinol. It was recently shown that by binding RBP, megalin has an essential role in transepithelial transport of retinol. It is possible that in vivo, megalin might also be responsible for the tubular resorption of TTR. To investigate this hypothesis we performed different TTR binding assays by surface plasmon ressonance (SPR) analysis and by using immortalized rat yolk sac cells with high expression levels of megalin. Binding of purified TTR, free as well as in complex with thyroxine or retinol, to immobilized megalin was shown by the SPR analysis. Radiolabeled TTR was rapidly taken up by cultured cells and the uptake was partially inhibited by a polyclonal megalin antibody and by the receptor-associated protein (RAP), a chaperone-like protein inhibiting ligand binding to megalin and other LDLr family receptors. The present data indicate that T'I'R represents a novel megalin ligand of potential importance in the transepithelial transport of retinol and thyroxine.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000