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Liver Δ6- and Δ9-desaturase activities in rats treated with peroxisome or mitochondria proliferators
Poster Presentations Wednesday 23 July
P177
EFA & Eicosanoids 1997 - Edinburgh
257
P178
Diabetes Alters Phospholipid and 1,2-Diacylglycerol Essential Fatty Acid Composition of Vascular Smooth Muscle. Pehowich, D.J., University of Alberta, Edmonton, 1.
Comparison of Different Oral Applications of ~3C Labelled Linoleic Acid H. Demmelmair, B. Wehner, A. Pfeiffer, B. Koletzko Kinderpoliklinik, Ludwig-Maximilians-Universit~it Mtinchen, Germany
Decreased response to vasoconstrictor hormones in diabetes may be associated with alterations in the molecular species of cellular 1,2diacylglycerol (1,2-DAG). This study was designed to determine how diabetes influences the essential fatty acid content of vascular smooth muscle 1,2-DAG, and its phospholipid precursors. Streptozotocininduced diabetic rats (n=8) and controls (n=8) were fed diets containing either 1% or 5% (w/w) (0-3 fatty acids for 4 weeks and then the fatty acid composition of thoracic aortae phospholipids and 1,2-DAG measured. In diabetic animals membrane levels of 18:2to-6 were elevated in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, whereas 20:4(0-6 was depleted, regardless of diet fed (p<0.01), indicating inhibition of A 6- and AS-desaturase activity. 1,2-DAG from diabetic rats fed the low (0-3 diet contained 56%+_2.0 more 18:26o-6 and 28%_+1.4 less 20:4(0-6 than controls fed the same diet (p<0.01). Feeding the high (0-3 diet increased 1,2DAG content of(0-3 fatty acids seven-fold at the expense of a further reduction in 20:460-6. When incubated with 100 gM angiotensin II, the contractile response of intact aortic rings from diabetic animals fed the high (0-3 diet was only 60.8%_+9.3 (p<0.01) that of nondiabetic animals fed the same diet. However, contractile response was not significantly different between diabetic animals fed the high (0-3 diet and controls fed the low o-3 diet (60.8%_+9.3 vs 55.6%_+7.9; p<0.05). The results indicate that vascular smooth muscle (0-6 and (0-3 fatty acid metabolism is altered in the diabetic state resulting in changes to the fatty acid profile of 1,2-DAG, and that enhancing eo-3 content partially ameliorates the depressed response to angiotensin II.
The endogenous conversion of linoleic acid (LA) into its long chain polyunsaturated derivatives (LCP), mainly arachidonic acid (AA), is important for infant nutrition. The contribution of body synthesis to available LCP in rive can be elucidated by using 13C-labelled tracers. Since blood sampling in infants needs to be minimised for ethical reasons, we tried to develop a protocol which requires only two blood samples and evaluated the information ttainable from a study in adults. Methods:. The oral tracer dose was 1 mg UJ3C-LA/kg BW. 5 subjects (62_+6 kg; M_+SD) received a bolus dose of tracer, while in the fraction group (n=4; 67_+3 kg) the tracer was split into 9 equal portions which were given during 3 days. In the bolus group blood was sampled before and 7 times during 7 d after tracer intake. In the fraction group samples were obtained before and 22 times during 10 d following the start of intake. Fatty acids from serum phospholipids were analysed for their concentration and 13C-content by GC-combustion-isotope ratio-MS. Areas under the tracer-concentrationtime curves (AUC) were calculated for LA, dihomo-7-1inolenic acid (DGLA) and AA. Using the SAAM II software fractional transfer rates (k) were estimated for the fatty acids. Results: The calculated parameters 'median) are shown in the table ~<0.05 between applications, [%/d] or [%] Bolus SD Frac. SD Mann-Whitney test, bp< kLA 93.7 12.9 80.0 3.3 0.05 k vs. AUC-ratio). kLA.DGLA 1.5a 0.6 2.1 a'D 0.4 AUC-ratios correlated kLA.AA 0.3 0.2 0.60 0.2 significantly with tracer AUCDcLA/AUCLa 4.1 1.4 5. I ° 0.8 concentrations at 48 h. AUCAA/AUCLA 1.7 0.8 2,6 ° 0.9 Conclusions: I. AUCDGLA/LA(48h) 6.3 3.0 2.8 0.3 ratios overestimate the AA/LA(48h) 1.5 l. 1 0.9 0.7 conversion of LA to LCP, because of differences in the turnover rates. 2. The difference between the application modes indicates that not all assumptions of the model are satisfied. 3. Even from a single blood sample after tracer.application, conversion can be estimated with a somewhat higher variability.
P179
P180
Plasma Triglyceride Lowering by Fish Oil: Docosahexaenoic Acid Metabolism in Rats Treated with Peroxisome or Mitochondrial Proliferators. Ming-Yi Chen, Hui-Min Su, Thomas N. Corse, and J. Thomas Brenna, Cornell University, Ithaca, NY, USA.
Liver A6- and A9-Desaturase Activities in Rats Treated With Peroxisome or Mitochondria Proliferators. Hui-Min Su, Ming-Yi Chen, and J. Thomas Brenna, Division o f Nutritional Sciences. Cornell University, Ithaca, NY 14853, USA
Mthough it is well known that intake o f dietary n-3 tong chain polyunsaturates (LCP(n-3)) result in lowering o f plasma triglycerides (TG), the mechanism is not well understood. Methods: We placed rats on a balanced control diet (C) with soybean oil as a source of fat, and treated groups as follows. F: LCP(n-3) from fish oil concentrate (30% EPA, 20% DHA), G: the peroxisome proliferator gemfibrozil, T: thyroxine (T4) i.v., a mitochondria profilerator which enhances energy expenditure, S: starvation. After several days of treatment, each animal was gavaged with 1 mg [U-13C]-DHA, and sacrificed 3 days later. Fatty acids were extracted from organs according to conventional procedures, with profiles quantified by capillary GC and analysed for isotopic composition by high precision mass spectrometry. Results: MI treatment groups had lower plasma TG than the controls (p<0.05). The liver DHA concentration in groups G, T, and F were 72%, 78%, and 134% o f the control value, respectively (p<0.05). The atom percent excess (APE, tracer:tracee) for liver DHA was greater than controls in the G and S groups but not different in the T group, while for EPA T was lower than C, which was not different from G The S group had the highest APE for both EPA and DHA suggesting LCP(n-3) are spared in starvation. We conclude that the TG-lower effect o f peroxisome-profilerators does not operate by increasing oxidation o f DHA, as may be the case for thyroxine.
The effect o f peroxisome and mitochondria proliferation on A6- and A9- desaturase activities and on lipid level in rat liver microsomes was studied with high precision mass spectrometry and stable isotopes. Five weeks old male Wistar rats were gavaged with Long chain polyunsaturate LCP(n-3) concentrate (30% EPA, 20% DHA, 0.375g/rat/day), gemfibrozil (a peroxisome proliferator, 50mg/kg wt) for two weeks, or injected with thyroxine (a mitochondrial profilerator, l mg/kg wt) for 5 days before sacrifice. Beside these treatments, all rats were fed fat-free chow and gavaged with 0.75g soybean oil for two weeks including a control group. A6-desaturase activity measured by the conversion o f [U-13C]-18:2n-6 to 18:3n-6 decreased in order o f groups: Thyroxine > Gemfibrozil > Control = LCP(n-3). A9-desaturase activity determined from the conversion o f [U-13C]-16:0 to 16:1n-7 decreased as Thyroxine > Control > Gemfibrozil > LCP(n-3) groups. The lipid level in liver microsomes decreased in order: Thyroxine (80p.g/mg rat liver microsome protein) > Gemfibrozil (150gg/mg) > Control (200gg/m) > LCP(n-3) (250/ag/mg) groups. Mthough LCP(n-3) group contained the highest lipid level per mg rat liver microsome protein, the LCPUFA group had the highest fraction of PUFA (43%). No significant difference was found in n-3 fatty acid profiles among the four groups. This study shows I) LCP(n-3) has little effect A6desaturase activity, 2) both peroxisomal and mitochondrial proliferation strongly influence desaturase activities.
P177
EFA & Eicosanoids 1997 - Edinburgh
257
P178
Diabetes Alters Phospholipid and 1,2-Diacylglycerol Essential Fatty Acid Composition of Vascular Smooth Muscle. Pehowich, D.J., University of Alberta, Edmonton, 1.
Comparison of Different Oral Applications of ~3C Labelled Linoleic Acid H. Demmelmair, B. Wehner, A. Pfeiffer, B. Koletzko Kinderpoliklinik, Ludwig-Maximilians-Universit~it Mtinchen, Germany
Decreased response to vasoconstrictor hormones in diabetes may be associated with alterations in the molecular species of cellular 1,2diacylglycerol (1,2-DAG). This study was designed to determine how diabetes influences the essential fatty acid content of vascular smooth muscle 1,2-DAG, and its phospholipid precursors. Streptozotocininduced diabetic rats (n=8) and controls (n=8) were fed diets containing either 1% or 5% (w/w) (0-3 fatty acids for 4 weeks and then the fatty acid composition of thoracic aortae phospholipids and 1,2-DAG measured. In diabetic animals membrane levels of 18:2to-6 were elevated in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, whereas 20:4(0-6 was depleted, regardless of diet fed (p<0.01), indicating inhibition of A 6- and AS-desaturase activity. 1,2-DAG from diabetic rats fed the low (0-3 diet contained 56%+_2.0 more 18:26o-6 and 28%_+1.4 less 20:4(0-6 than controls fed the same diet (p<0.01). Feeding the high (0-3 diet increased 1,2DAG content of(0-3 fatty acids seven-fold at the expense of a further reduction in 20:460-6. When incubated with 100 gM angiotensin II, the contractile response of intact aortic rings from diabetic animals fed the high (0-3 diet was only 60.8%_+9.3 (p<0.01) that of nondiabetic animals fed the same diet. However, contractile response was not significantly different between diabetic animals fed the high (0-3 diet and controls fed the low o-3 diet (60.8%_+9.3 vs 55.6%_+7.9; p<0.05). The results indicate that vascular smooth muscle (0-6 and (0-3 fatty acid metabolism is altered in the diabetic state resulting in changes to the fatty acid profile of 1,2-DAG, and that enhancing eo-3 content partially ameliorates the depressed response to angiotensin II.
The endogenous conversion of linoleic acid (LA) into its long chain polyunsaturated derivatives (LCP), mainly arachidonic acid (AA), is important for infant nutrition. The contribution of body synthesis to available LCP in rive can be elucidated by using 13C-labelled tracers. Since blood sampling in infants needs to be minimised for ethical reasons, we tried to develop a protocol which requires only two blood samples and evaluated the information ttainable from a study in adults. Methods:. The oral tracer dose was 1 mg UJ3C-LA/kg BW. 5 subjects (62_+6 kg; M_+SD) received a bolus dose of tracer, while in the fraction group (n=4; 67_+3 kg) the tracer was split into 9 equal portions which were given during 3 days. In the bolus group blood was sampled before and 7 times during 7 d after tracer intake. In the fraction group samples were obtained before and 22 times during 10 d following the start of intake. Fatty acids from serum phospholipids were analysed for their concentration and 13C-content by GC-combustion-isotope ratio-MS. Areas under the tracer-concentrationtime curves (AUC) were calculated for LA, dihomo-7-1inolenic acid (DGLA) and AA. Using the SAAM II software fractional transfer rates (k) were estimated for the fatty acids. Results: The calculated parameters 'median) are shown in the table ~<0.05 between applications, [%/d] or [%] Bolus SD Frac. SD Mann-Whitney test, bp< kLA 93.7 12.9 80.0 3.3 0.05 k vs. AUC-ratio). kLA.DGLA 1.5a 0.6 2.1 a'D 0.4 AUC-ratios correlated kLA.AA 0.3 0.2 0.60 0.2 significantly with tracer AUCDcLA/AUCLa 4.1 1.4 5. I ° 0.8 concentrations at 48 h. AUCAA/AUCLA 1.7 0.8 2,6 ° 0.9 Conclusions: I. AUCDGLA/LA(48h) 6.3 3.0 2.8 0.3 ratios overestimate the AA/LA(48h) 1.5 l. 1 0.9 0.7 conversion of LA to LCP, because of differences in the turnover rates. 2. The difference between the application modes indicates that not all assumptions of the model are satisfied. 3. Even from a single blood sample after tracer.application, conversion can be estimated with a somewhat higher variability.
P179
P180
Plasma Triglyceride Lowering by Fish Oil: Docosahexaenoic Acid Metabolism in Rats Treated with Peroxisome or Mitochondrial Proliferators. Ming-Yi Chen, Hui-Min Su, Thomas N. Corse, and J. Thomas Brenna, Cornell University, Ithaca, NY, USA.
Liver A6- and A9-Desaturase Activities in Rats Treated With Peroxisome or Mitochondria Proliferators. Hui-Min Su, Ming-Yi Chen, and J. Thomas Brenna, Division o f Nutritional Sciences. Cornell University, Ithaca, NY 14853, USA
Mthough it is well known that intake o f dietary n-3 tong chain polyunsaturates (LCP(n-3)) result in lowering o f plasma triglycerides (TG), the mechanism is not well understood. Methods: We placed rats on a balanced control diet (C) with soybean oil as a source of fat, and treated groups as follows. F: LCP(n-3) from fish oil concentrate (30% EPA, 20% DHA), G: the peroxisome proliferator gemfibrozil, T: thyroxine (T4) i.v., a mitochondria profilerator which enhances energy expenditure, S: starvation. After several days of treatment, each animal was gavaged with 1 mg [U-13C]-DHA, and sacrificed 3 days later. Fatty acids were extracted from organs according to conventional procedures, with profiles quantified by capillary GC and analysed for isotopic composition by high precision mass spectrometry. Results: MI treatment groups had lower plasma TG than the controls (p<0.05). The liver DHA concentration in groups G, T, and F were 72%, 78%, and 134% o f the control value, respectively (p<0.05). The atom percent excess (APE, tracer:tracee) for liver DHA was greater than controls in the G and S groups but not different in the T group, while for EPA T was lower than C, which was not different from G The S group had the highest APE for both EPA and DHA suggesting LCP(n-3) are spared in starvation. We conclude that the TG-lower effect o f peroxisome-profilerators does not operate by increasing oxidation o f DHA, as may be the case for thyroxine.
The effect o f peroxisome and mitochondria proliferation on A6- and A9- desaturase activities and on lipid level in rat liver microsomes was studied with high precision mass spectrometry and stable isotopes. Five weeks old male Wistar rats were gavaged with Long chain polyunsaturate LCP(n-3) concentrate (30% EPA, 20% DHA, 0.375g/rat/day), gemfibrozil (a peroxisome proliferator, 50mg/kg wt) for two weeks, or injected with thyroxine (a mitochondrial profilerator, l mg/kg wt) for 5 days before sacrifice. Beside these treatments, all rats were fed fat-free chow and gavaged with 0.75g soybean oil for two weeks including a control group. A6-desaturase activity measured by the conversion o f [U-13C]-18:2n-6 to 18:3n-6 decreased in order o f groups: Thyroxine > Gemfibrozil > Control = LCP(n-3). A9-desaturase activity determined from the conversion o f [U-13C]-16:0 to 16:1n-7 decreased as Thyroxine > Control > Gemfibrozil > LCP(n-3) groups. The lipid level in liver microsomes decreased in order: Thyroxine (80p.g/mg rat liver microsome protein) > Gemfibrozil (150gg/mg) > Control (200gg/m) > LCP(n-3) (250/ag/mg) groups. Mthough LCP(n-3) group contained the highest lipid level per mg rat liver microsome protein, the LCPUFA group had the highest fraction of PUFA (43%). No significant difference was found in n-3 fatty acid profiles among the four groups. This study shows I) LCP(n-3) has little effect A6desaturase activity, 2) both peroxisomal and mitochondrial proliferation strongly influence desaturase activities.