Oxidation rates in WT and VLCAD^−/− mice fed either with LCT or MCT diet.

<p>Oxidation rate was determined with straight-chain acyl-CoA substrates. Tissues tested were (A) liver, (B) heart and (C) skeletal muscle. Oxidation is expressed in mU/mg protein and is given as mean of mean ± SEM (<i>n</i> = 5–7).* indicates significant differences between WT and VLCAD^−/− mice within a diet group. # indicates significant differences between WT or VLCAD^−/− mice under different dietary conditions. Values are considered significantly different if p<0.05 (Two way ANOVA and Student's t-test).</p

Mean body weight of WT and VLCAD^−/− mice fed either with LCT or MCT diet.

<p>Body weights are expressed in gram (g) and are given as mean of mean ± SEM (<i>n</i> = 5–7).</p

Tissue-Specific Strategies of the Very-Long Chain Acyl-CoA Dehydrogenase-Deficient (VLCAD^−/−) Mouse to Compensate a Defective Fatty Acid β-Oxidation

<div><p>Very long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency is the most common long-chain fatty acid oxidation disorder presenting with heterogeneous phenotypes. Similar to many patients with VLCADD, VLCAD-deficient mice (VLCAD^−/−) remain asymptomatic over a long period of time. In order to identify the involved compensatory mechanisms, wild-type and VLCAD^−/− mice were fed one year either with a normal diet or with a diet in which medium-chain triglycerides (MCT) replaced long-chain triglycerides, as approved intervention in VLCADD. The expression of the mitochondrial long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) was quantified at mRNA and protein level in heart, liver and skeletal muscle. The oxidation capacity of the different tissues was measured by LC-MS/MS using acyl-CoA substrates with a chain length of 8 to 20 carbons. Moreover, in white skeletal muscle the role of glycolysis and concomitant muscle fibre adaptation was investigated. In one year old VLCAD^−/− mice MCAD and LCAD play an important role in order to compensate deficiency of VLCAD especially in the heart and in the liver. However, the white gastrocnemius muscle develops alternative compensatory mechanism based on a different substrate selection and increased glucose oxidation. Finally, the application of an MCT diet over one year has no effects on LCAD or MCAD expression. MCT results in the VLCAD^−/− mice only in a very modest improvement of medium-chain acyl-CoA oxidation capacity restricted to cardiac tissue. In conclusion, VLCAD^−/− mice develop tissue-specific strategies to compensate deficiency of VLCAD either by induction of other mitochondrial acyl-CoA dehydrogenases or by enhancement of glucose oxidation. In the muscle, there is evidence of a muscle fibre type adaptation with a predominance of glycolytic muscle fibres. Dietary modification as represented by an MCT-diet does not improve these strategies long-term.</p> </div

mRNA expression of different dehydrogenases in mice fed either with an LCT or MCT diet.

<p>LCT, long-chain triglyceride; MCT, medium-chain triglyceride; <i>ACADM</i>, medium-chain acyl-CoA dehydrogenase and <i>ACADL,</i> long-chain acyl-CoA dehydrogenase.Values are mean of mean ± SEM (<i>n</i> = 5–7). * indicates significant differences between WT and VLCAD^−/− mice within a diet group. # indicates significant differences between WT or VLCAD^−/− mice under different dietary conditions. Values are considered significantly different if p<0.05 (Two way ANOVA and Student's t-test).</p

Functional Effects of Different Medium-Chain Acyl-CoA Dehydrogenase Genotypes and Identification of Asymptomatic Variants

<div><p>Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (OMIM 201450) is the most common inherited disorder of fatty acid metabolism presenting with hypoglycaemia, hepatopathy and Reye-like symptoms during catabolism. In the past, the majority of patients carried the prevalent c.985A>G mutation in the <em>ACADM</em> gene. Since the introduction of newborn screening many other mutations with unknown clinical relevance have been identified in asymptomatic newborns. In order to identify functional effects of these mutant genotypes we correlated residual MCAD (OMIM 607008) activities as measured by octanoyl-CoA oxidation in lymphocytes with both genotype and relevant medical reports in 65 newborns harbouring mutant alleles. We identified true disease-causing mutations with residual activities of 0 to 20%. In individuals carrying the c.199T>C or c.127G>A mutation on one allele, residual activities were much higher and in the range of heterozygotes (31%–60%). Therefore, both mutations cannot clearly be associated with a clinical phenotype. This demonstrates a correlation between the octanoyl-CoA oxidation rate in lymphocytes and the clinical outcome. With newborn screening, the natural course of disease is difficult to assess. The octanoyl-CoA oxidation rate, therefore, allows a risk assessment at birth and the identification of new <em>ACADM</em> genotypes associated with asymptomatic disease variants.</p> </div

Western blot analysis of different dehydrogenases in mice fed either with LCT or MCT diet.

<p>LCT, long-chain triglyceride; MCT, medium-chain triglyceride; <i>ACADM</i>, medium-chain acyl-CoA dehydrogenase; <i>ACADL,</i> long-chain acyl-CoA dehydrogenase; <i>GAPDH,</i> Glyceraldehyde 3-phosphate dehydrogenase. Each <i>lane</i> represents pooled homogenates and contains 10 µg of liver / heart lysates and 20 µg of gastrocnemius muscle. Pools are prepared with protein homogenates of <i>n</i> = 5–7 animals.</p

Correlation of octanoyl-CoA oxidation residual activities [%] detected by HPLC-UV and by HPLC-ESI-MS/MS.

<p>Relative residual octanoyl-CoA oxidation activities are presented as a percentage of the mean value of healthy controls. The resulting regression line is y = 1.16×+0.03. The correlation coefficient R² is 0.96.</p

Correlation of octanoyl-CoA oxidation with <i>ACADM</i> genotype.

<p>The patients are grouped into 1. Wild-types, 2. Individuals with only one mutation found and 3. Homozygous and compound heterozygous newborns. Patients with identical genotypes are grouped in one bar. Homozygotes and compound heterozygotes are shown in black, heterozygotes in shaded and wild-types in white bars. * denotes novel identified missense mutations. Relative residual octanoyl-CoA oxidation activities are presented as a percentage of the mean value of healthy controls ± standard error of the mean (SEM).</p

Substrate selection and gene expression of different muscle fibre types.

<p>LCT, long-chain triglyceride; MCT, medium-chain triglyceride; <i>PGC1a,</i> peroxisome proliferative activated receptor gamma coactivator 1 alpha; <i>PDK4,</i> pyruvate dehydrogenase kinase isoenzyme 4; <i>Tnni1,</i> troponin I skeletal, slow 1; <i>Tnni2,</i> troponin I, skeletal, fast 2; <i>Tnnc1,</i> troponin C, cardiac/slow skeletal; <i>Tnnc2</i>, troponin C2, fast; <i>Tnnt1</i>, troponin T1, skeletal, slow. Values are mean of mean ± SEM (<i>n</i> = 5–7). * indicates significant differences between WT and VLCAD^−/− mice within a diet group. # indicates significant differences between WT or VLCAD^−/− mice under different dietary conditions. Values are considered significant if p<0.05 (Two way ANOVA and Student's t-test).</p

Primer used for RT-PCR analysis.

<p><i>ACADM,</i> medium-chain acyl-CoA dehydrogenase; <i>ACADL,</i> long-chain acyl-CoA dehydrogenase; <i>PGC1a,</i> peroxisome proliferative activated receptor gamma coactivator 1 alpha; <i>PDK4,</i> pyruvate dehydrogenase kinase isoenzyme 4; <i>PPARa,</i> peroxisome proliferative activated receptor alpha; <i>Tnni1</i>, troponin I skeletal, slow 1; <i>Tnni2</i>, troponin I, skeletal, fast 2; <i>Tnnc1</i>, troponin C, cardiac/slow skeletal ; <i>Tnnc2</i>, troponin C2, fast and <i>Tnnt1,</i> troponin T1, skeletal, slow.</p