Altered carnitine homeostasis is associated with decreased mitochondrial function and altered nitric oxide signaling in lambs with pulmonary hypertension
Shruti SharmaNeetu SudDean A. WisemanA. Lee CarterSanjiv KumarYali HouThomas RauJason M. WilhamCynthia HarmonPeter OishiJeffrey R. FinemanStephen M. Black
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Abstract:
Utilizing aortopulmonary vascular graft placement in the fetal lamb, we have developed a model (shunt) of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. Our previous studies have identified a progressive development of endothelial dysfunction in shunt lambs that is dependent, at least in part, on decreased nitric oxide (NO) signaling. The purpose of this study was to evaluate the possible role of a disruption in carnitine metabolism in shunt lambs and to determine the effect on NO signaling. Our data indicate that at 2 wk of age, shunt lambs have significantly reduced expression ( P < 0.05) of the key enzymes in carnitine metabolism: carnitine palmitoyltransferases 1 and 2 as well as carnitine acetyltransferase (CrAT). In addition, we found that CrAT activity was inhibited due to increased nitration. Furthermore, free carnitine levels were significantly decreased whereas acylcarnitine levels were significantly higher in shunt lambs ( P < 0.05). We also found that alterations in carnitine metabolism resulted in mitochondrial dysfunction, since shunt lambs had significantly decreased pyruvate, increased lactate, and a reduced pyruvate/lactate ratio. In pulmonary arterial endothelial cells cultured from juvenile lambs, we found that mild uncoupling of the mitochondria led to a decrease in cellular ATP levels and a reduction in both endothelial NO synthase-heat shock protein 90 (eNOS-HSP90) interactions and NO signaling. Similarly, in shunt lambs we found a loss of eNOS-HSP90 interactions that correlated with a progressive decrease in NO signaling. Our data suggest that mitochondrial dysfunction may play a role in the development of endothelial dysfunction and pulmonary hypertension and increased pulmonary blood flow.Keywords:
Homeostasis
Carnitine O-palmitoyltransferase
Carnitine O-palmitoyltransferase
Metabolic disorder
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Carnitine O-palmitoyltransferase
Carnitine palmitoyltransferase I
Microsoma
Transferase
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Carnitine O-palmitoyltransferase
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Translocase
Carnitine O-palmitoyltransferase
Sarcolemma
Carnitine palmitoyltransferase I
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Carnitine O-palmitoyltransferase
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Carnitine O-palmitoyltransferase
Carnitine palmitoyltransferase I
Anaerobic glycolysis
Carbohydrate Metabolism
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Recently a number of defects of fatty acid transport and beta-oxidation have been described. Primary carnitine deficiency presents itself as muscle carnitine deficiency, systemic carnitine deficiency or a cardiomyopathic subtype. Another abnormality of fatty acid transport is due to inner carnitine palmitoyl transferase deficiency, usually associated with a myoglobinuric phenotype. Long chain, medium and short chain acyl-CoA-dehydrogenase defects are usually associated to Reye's syndrome, encephalopathy, organic aciduria and secondary carnitine deficiency.
Carnitine O-palmitoyltransferase
Reye Syndrome
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OBJECTIVES: Carnitine palmitoyltransferase (CPT) deficiency is one of the most common defects of mitochondrial fatty acid oxidation. Two different enzymes (CPT-I and CPT-II) are involved. Due to problems in measuring enzyme activity, relatively little is known about the substrate specificity of each of the human enzymes. This is of considerable importance in the treatment of patients. The objectives were to establish a reliable method for the measurement of CPT activity in whole cells, to use this to characterise the substrate specificity of each enzyme, and finally, to determine if medium chain triglycerides would be of benefit in the treatment of deficient patients. METHODS: A simple permeabilisation technique was used which allows the measurement of CPT activity in a small amount of cultured skin fibroblasts or peripheral blood cells. Using this technique three patients were identified with CPT deficiency. In two of these patients, one with CPT-I deficiency and one with CPT-II deficiency, a complete substrate specificity profile of the mitochondrial carnitine acyltransferases was established for all saturated even chain acyl-CoA esters. RESULTS: For both enzymes the highest CPT activity was with C12-CoA. About 70% of total cellular carnitine octanoyltransferase activity was due to mitochondrial CPT. As CPT is involved in the transport of medium chain fatty acids the metabolic response of a patient with CPT-II deficiency to dietary medium chain triglycerides was assessed. Despite the normal production of ketone bodies there was a significant medium chain dicarboxylic aciduria in the patient, indicating a limited capacity of the CPT independent mitochondrial uptake of medium chain fatty acids. CONCLUSIONS: CPT deficiency can easily be diagnosed in permeabilised cultured skin fibroblasts. Both CPT-I and CPT-II are more active with medium chain length substrates than previously assumed. Care should therefore be taken in the treatment of these patients with medium chain triglycerides.
Carnitine O-palmitoyltransferase
Carnitine palmitoyltransferase I
Acyltransferases
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The activities of carnitine palmitoyltransferases (CPTs) of mitochondrial outer and inner membranes and of peroxisomes have been studied with carnitine analogues, namely DL-thiolcarnitine, DL-sulphocarnitine and L-aminocarnitine, using palmitoyl-CoA or octanoyl-CoA as co-substrate. With sulphocarnitine, both of the mitochondrial CPTs and the malonyl-CoA-sensitive CPT of peroxisomes showed appreciable activity with palmitoyl-CoA, but relatively lower activity when octanoyl-CoA was the co-substrate. The soluble CPT of peroxisomes did not show any activity with sulphocarnitine in the presence of either acyl-CoA. With thiolcarnitine, all of the CPTs showed more activity with palmitoyl-CoA than with octanoyl-CoA. None of the CPTs showed any activity with aminocarnitine and palmitoyl-CoA, but when the acyl donor was octanoyl-CoA, both of the malonyl-CoA-sensitive CPT enzymes showed considerable activity, unlike the malonyl-CoA-insensitive CPT isoenzymes. Aminocarnitine inhibited palmitoylcarnitine formation by both of the mitochondrial CPTs and by the CPT of gradient-purified peroxisomes, but the purified peroxisomal soluble CPT was not inhibited. These results show that the interaction of CPT enzymes with carnitine analogues, as substrates or inhibitors, is influenced by the chain length of the acyl-CoA substrate, and that the use of the appropriate carnitine analogue and acyl-CoA is likely to be useful for the discrimination of the various CPT activities in CPT deficiency disorders.
Palmitoylcarnitine
Carnitine O-palmitoyltransferase
Carnitine palmitoyltransferase I
Acyl-CoA
Malonyl-CoA
Coenzyme A
Acetyl-CoA
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瞄准：到可诱导的氮的氧化物 synthase (i NOS ) 和在有导致乙醇的肝损伤的老鼠和他们有肝的关系的内皮的氮的氧化物 synthase (eNOS ) 的表示和活动损坏的学习，原子 factor-kappaB (NF-kappaB ) 和肿瘤坏死 factor-alpha (TNF-alpha ) 的激活在肝的表示。方法：女 Sprague-Dawley 老鼠为 4 或 6 wk 经由胃管饲法与乙醇或 isocaloric 葡萄糖日报一起被给鱼油(0.5 mL ) 。肝损伤用浆液丙氨酸 aminotransferase (中高音) 被估计活动和病理学的分析。肝 malondialdehyde (MDA ) ，氮的氧化物内容， i NOS 和 eNOS 活动是坚定的。在肝的 NF-kappaB p65iiNOS， eNOS 和 TNF-alpha 蛋白质或 mRNA 表示被免疫组织化学或反向的 transcriptase 聚合酶链反应(RT-PCR ) 检测。结果：为 4 wk 的长期的乙醇管饲法在肝引起了脂肪变性，发炎和坏死，并且提高了浆液中高音活动。延长乙醇管理(6 wk ) 提高了肝损坏。这些回答每氧化，没有内容， i NOS 活动和减少的 eNOS 活动伴有增加的类脂化合物。NF-kappaB p65， i NOS 和 TNF-alpha 蛋白质或 mRNA 表示显著地在长期的乙醇管饲法以后被导致，而 eNOS mRNA 表示仍然保持未改变。提高的 i NOS 活动和表示断然与肝损坏，特别 necro 发炎， NF-kappaB 的激活，和 TNF-alpha mRNA 表示被相关。结论：i NOS 表示和活动在老鼠在长期的乙醇暴露以后在肝被导致，它与肝损坏，特别 necro 发炎， NF-kappaB 的激活和 TNF-alpha 表示被相关。eNOS 活动被减少，但是它的 mRNA 表示没被影响。
Malondialdehyde
Endothelial NOS
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