- Email: [email protected]
Isolation and characterization of mouse lipoxygenase genes
$66
Poster session abstracts / A therosclerosis 115 (Suppl.) (1995) $45-$129 P6 Animal Models
242
244
CLUSTERING OF ALTERED GLUCOREGULATION, HYPERTENSION, AND LIPOPEROXIDATION IN HEREDITARY HYPERTRIGLYCERIDEMIC RATS A. Vr~na, L. Kazdov~i, Z. Dobe~ov~t, M. Mat~:j~kov~i, J. Kune~ Institute for Clinical and Experimental Medicine, Prague, Czech Republic
PLASMA TR1GLYCERIDE METABOLISM IN APOLIPOPROTEIN-E'3-LEIDEN TRANSGENIC MICE M.C. Jong, V.E.H. Dahlmans, B.J.M. van Vlijmen, M. Breuer, R R Frants, M.H. Hofker. L.M. Havekes TNO-PG, Gaubius labarotory and MGC-Department of Human Genetics, Leiden, The Netherlands
A new experimental model of hypertriglyceridemia, hereditary hypertriglyceridemic (HTg) rats, was recently developed in our laboratory. In addition to distinctly increased triglyceridemia, especially during high carbohydrate intake, these rats exhibited the following alterations of glucoregulation, blood pressure and lipid peroxidation: 1. Oral glucose tolerance in these rats was impaired compared with controls, while serum insulin was higher indicating a decrease in insulin action. Glucose uptake measured using in vivo accumulation of 2-deoxyglucose was lower both in muscle and in adipose tissue of HTg rats. 2. Systolic (SBP) and diastolic (DBP) blood pressure (mm Hg) were higher in HTg rats as compared with controls (SBP: 156 ± 2 vs 126 ± 3, DBP: 110 ± 2 vs 88 ± 2, p < 0.001). 3. Lipid peroxides measured as conjugated dienes (CD) or malondialdehyde (MDA) in serum and in the liver were higher in HTg rats as compared with controls (serum CD: 0 . 2 8 ± 0 . 0 2 vs 0.18_+0.01 U, p < 0.02, MDA: 2.12±0.02 vs 1.75_+0.01 nmol/ml, p < 0.05, liver MDA: 11.03±0.4 vs 9.72±0.2 nmol/g, p < 0.05). Conclusion: 1.The HTg line, exhibiting a cluster of symptoms of metabolic syndrome X (hypertriglyeeridemia, insulinresistance, hyperinsulinemia, elevated blood pressure) could be regarded as a model of choice to study the pathophysiology of the Syndrome and to develop a strategy of its management. 2. In addition, this is the first demonstration indicating that metabolic disorders of Syndrome X are accompanied by adverse alterations of lipoperoxidation.
In previous work APOE'3-Leiden transgenic mice (line #2) showed hyperlipoproteinemia upon feeding high fat high cholesterol diet (HFC). These mice carry, in addition to the APOE*3-Leiden gene, also the human APOC1 gene and the hepatic control region (HCR). To investigate the possible effect of the simultaneous expression of the human APOC1 gene on the phenotypic expression, we generated transgenic mice carrying an APOE*3-Leiden-HCR construct, thus without the APOCI gene (line #10.1). Both lines were similar in exhibiting hypercholesterolemia on HFC (6.3 and 7.1 nnnol/l for #10.1 and #2 respectively). However on low fat low cholesterol diet (LFC) APOE*3-Leiden-HCR female mice did not exhibit hypertriglyceridemia as did female mice of #2 (1.39 vs 4.3 mmol/I, respectively). To study plasma triglyceride metabolism, the hepatic VLDL production rate was measured after injection of Triton WRl339. The VLDL production rate in females of #10.1 was not different from controls (0.17 versus 0.14 mmol TG/hour/kg), whereas females of #2 exhibited a production rate of 0.23 nnnol TG/hour/kg. Furthermore lipolysis of VLDL ( d < l . 0 0 6 ) from females of #2 by lipoprotein lipase was less efficient than lipolysis of females of #10.1 VLDL (k,~=l.07 vs k~=0.32 mM Triglyceride). We therefore speculate that the hypertriglyceridemia observed in APOE'3-keiden mice expressing human APOC1 on LFC diet, might be due to enhanced triglyceride synthesis and disturbed lipolysis.
243
245
ISOLATION AND CHARACTERIZATION OF MOUSE LIPOXYGENASE GENES K. Willems van Dijk, K. Steketee, L.M. Havekes, R.R. Frants, M.H. Hofker MGC-Department of Human Genetics, Leiden University, and PGTNO, Gaubius Laboratory, Leiden, The Netherlands
THE EFFECT OF HYPERCHGLESTEROLEMIC DIET AND D2 VITAMIN ON THE ATHEROSCLEROSIS DEVELOPMENT 1N SAND RAT (PSAMMOMYS OBESUS) OBESE AND PREDIABETIC N.Bennani-Kabchi M.A. Bellabas, O. Lemsafre G. Marquie Unite nutrition, Faculte des Science, B.P.1014, Rabat, Marocco
An early event in the development of atherosclerosis is the accumulation of lipid-laden foam cells in the intima. Foam cells are thought to result from the unrestricted uptake of modified lipoproteins by macrophages. The enzyme 15-1ipoxygenase has been implicated in the oxidative modification of lipoproteins. To study the role of lipoxygenases in the atherosclerotic process in transgenic mouse models, we are cloning and characterizing mouse lipoxygenase genes. The screening of a genomic phage library with a human I5lipoxygenase cDNA probe resulted in two clones, phage#5 and phage#6. The phage#5 gene was sequenced completely and shown to consist of 14 exons spanning approximately 8 kb. The phage#6 gene was sequenced partly. In addition, the complete cDNA from the phage#5 gene and a 5-prime cDNA fragment from the phage#6 gene was cloned from reverse transcribed macrophage RNA and sequenced. The phage#6 gene is identical to the leukocyte-type 12-1ipoxygenase, recently described by Chen et al (J. Biol. Chem. 269:13979). A 12-1ipoxygenase related pseudogene described by these authors seems identical to the phage#5 gene. However, since we have found no indications for pseudogene status, we are currently in the process of determining the specificity and expression pattern of the phage#5 lipoxygenase.
Limited studies have been focussed on proving that the Sand rat (Psammomys Obesus) as a model of vascular pathology studies by means of hypercholesterolemic and atherogenic diet. Those studies came across the atheroresistance of the species. To that end, and due to the lack of studies on Moroccan Sand rat, we have experimentally reproduced atherogenic lesions by hypercholesterolemic diet associated with high proportions of D2 vitamin, during 2 months. Our results showed during the treatment, the development of obesity, prediabetes with dyslipidemy (hypercholesterolemy, hypertriglyceridemy, increase of LDL cholesterol and stability of HDL cholesterol (an antiatherogenic parameter). The pancreas showed hypertrophy and hyperplasy of Langerhans islands. We didn't observe the microangiopathy at the skin level and nor evident lipidic deposits at the liver during 2 months of treatment. The hypercaloric diet produced by itself, arterial preatheromateous lesions at the heart level. The addition of an oral treatment by D2 vitamin at high proportions, produced more important atherosclerosis lesions at various stages: lipidic, calcified, ulcerate, in aortic and renal artery with ischemic effects represented by myocardic infarctions.
Poster session abstracts / A therosclerosis 115 (Suppl.) (1995) $45-$129 P6 Animal Models
242
244
CLUSTERING OF ALTERED GLUCOREGULATION, HYPERTENSION, AND LIPOPEROXIDATION IN HEREDITARY HYPERTRIGLYCERIDEMIC RATS A. Vr~na, L. Kazdov~i, Z. Dobe~ov~t, M. Mat~:j~kov~i, J. Kune~ Institute for Clinical and Experimental Medicine, Prague, Czech Republic
PLASMA TR1GLYCERIDE METABOLISM IN APOLIPOPROTEIN-E'3-LEIDEN TRANSGENIC MICE M.C. Jong, V.E.H. Dahlmans, B.J.M. van Vlijmen, M. Breuer, R R Frants, M.H. Hofker. L.M. Havekes TNO-PG, Gaubius labarotory and MGC-Department of Human Genetics, Leiden, The Netherlands
A new experimental model of hypertriglyceridemia, hereditary hypertriglyceridemic (HTg) rats, was recently developed in our laboratory. In addition to distinctly increased triglyceridemia, especially during high carbohydrate intake, these rats exhibited the following alterations of glucoregulation, blood pressure and lipid peroxidation: 1. Oral glucose tolerance in these rats was impaired compared with controls, while serum insulin was higher indicating a decrease in insulin action. Glucose uptake measured using in vivo accumulation of 2-deoxyglucose was lower both in muscle and in adipose tissue of HTg rats. 2. Systolic (SBP) and diastolic (DBP) blood pressure (mm Hg) were higher in HTg rats as compared with controls (SBP: 156 ± 2 vs 126 ± 3, DBP: 110 ± 2 vs 88 ± 2, p < 0.001). 3. Lipid peroxides measured as conjugated dienes (CD) or malondialdehyde (MDA) in serum and in the liver were higher in HTg rats as compared with controls (serum CD: 0 . 2 8 ± 0 . 0 2 vs 0.18_+0.01 U, p < 0.02, MDA: 2.12±0.02 vs 1.75_+0.01 nmol/ml, p < 0.05, liver MDA: 11.03±0.4 vs 9.72±0.2 nmol/g, p < 0.05). Conclusion: 1.The HTg line, exhibiting a cluster of symptoms of metabolic syndrome X (hypertriglyeeridemia, insulinresistance, hyperinsulinemia, elevated blood pressure) could be regarded as a model of choice to study the pathophysiology of the Syndrome and to develop a strategy of its management. 2. In addition, this is the first demonstration indicating that metabolic disorders of Syndrome X are accompanied by adverse alterations of lipoperoxidation.
In previous work APOE'3-Leiden transgenic mice (line #2) showed hyperlipoproteinemia upon feeding high fat high cholesterol diet (HFC). These mice carry, in addition to the APOE*3-Leiden gene, also the human APOC1 gene and the hepatic control region (HCR). To investigate the possible effect of the simultaneous expression of the human APOC1 gene on the phenotypic expression, we generated transgenic mice carrying an APOE*3-Leiden-HCR construct, thus without the APOCI gene (line #10.1). Both lines were similar in exhibiting hypercholesterolemia on HFC (6.3 and 7.1 nnnol/l for #10.1 and #2 respectively). However on low fat low cholesterol diet (LFC) APOE*3-Leiden-HCR female mice did not exhibit hypertriglyceridemia as did female mice of #2 (1.39 vs 4.3 mmol/I, respectively). To study plasma triglyceride metabolism, the hepatic VLDL production rate was measured after injection of Triton WRl339. The VLDL production rate in females of #10.1 was not different from controls (0.17 versus 0.14 mmol TG/hour/kg), whereas females of #2 exhibited a production rate of 0.23 nnnol TG/hour/kg. Furthermore lipolysis of VLDL ( d < l . 0 0 6 ) from females of #2 by lipoprotein lipase was less efficient than lipolysis of females of #10.1 VLDL (k,~=l.07 vs k~=0.32 mM Triglyceride). We therefore speculate that the hypertriglyceridemia observed in APOE'3-keiden mice expressing human APOC1 on LFC diet, might be due to enhanced triglyceride synthesis and disturbed lipolysis.
243
245
ISOLATION AND CHARACTERIZATION OF MOUSE LIPOXYGENASE GENES K. Willems van Dijk, K. Steketee, L.M. Havekes, R.R. Frants, M.H. Hofker MGC-Department of Human Genetics, Leiden University, and PGTNO, Gaubius Laboratory, Leiden, The Netherlands
THE EFFECT OF HYPERCHGLESTEROLEMIC DIET AND D2 VITAMIN ON THE ATHEROSCLEROSIS DEVELOPMENT 1N SAND RAT (PSAMMOMYS OBESUS) OBESE AND PREDIABETIC N.Bennani-Kabchi M.A. Bellabas, O. Lemsafre G. Marquie Unite nutrition, Faculte des Science, B.P.1014, Rabat, Marocco
An early event in the development of atherosclerosis is the accumulation of lipid-laden foam cells in the intima. Foam cells are thought to result from the unrestricted uptake of modified lipoproteins by macrophages. The enzyme 15-1ipoxygenase has been implicated in the oxidative modification of lipoproteins. To study the role of lipoxygenases in the atherosclerotic process in transgenic mouse models, we are cloning and characterizing mouse lipoxygenase genes. The screening of a genomic phage library with a human I5lipoxygenase cDNA probe resulted in two clones, phage#5 and phage#6. The phage#5 gene was sequenced completely and shown to consist of 14 exons spanning approximately 8 kb. The phage#6 gene was sequenced partly. In addition, the complete cDNA from the phage#5 gene and a 5-prime cDNA fragment from the phage#6 gene was cloned from reverse transcribed macrophage RNA and sequenced. The phage#6 gene is identical to the leukocyte-type 12-1ipoxygenase, recently described by Chen et al (J. Biol. Chem. 269:13979). A 12-1ipoxygenase related pseudogene described by these authors seems identical to the phage#5 gene. However, since we have found no indications for pseudogene status, we are currently in the process of determining the specificity and expression pattern of the phage#5 lipoxygenase.
Limited studies have been focussed on proving that the Sand rat (Psammomys Obesus) as a model of vascular pathology studies by means of hypercholesterolemic and atherogenic diet. Those studies came across the atheroresistance of the species. To that end, and due to the lack of studies on Moroccan Sand rat, we have experimentally reproduced atherogenic lesions by hypercholesterolemic diet associated with high proportions of D2 vitamin, during 2 months. Our results showed during the treatment, the development of obesity, prediabetes with dyslipidemy (hypercholesterolemy, hypertriglyceridemy, increase of LDL cholesterol and stability of HDL cholesterol (an antiatherogenic parameter). The pancreas showed hypertrophy and hyperplasy of Langerhans islands. We didn't observe the microangiopathy at the skin level and nor evident lipidic deposits at the liver during 2 months of treatment. The hypercaloric diet produced by itself, arterial preatheromateous lesions at the heart level. The addition of an oral treatment by D2 vitamin at high proportions, produced more important atherosclerosis lesions at various stages: lipidic, calcified, ulcerate, in aortic and renal artery with ischemic effects represented by myocardic infarctions.