Hiroko Nikaido

Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter

Primary systemic carnitine deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia. SCD has also been linked to sudden infant death syndrome. Membrane-physiological studies have suggested a defect of the carnitine transport system in the plasma membrane in SCD patients and in the mouse model, juvenile visceral steatosis (jvs; ref. 6). Although the responsible loci have been mapped in both human and mouse, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2 (ref. 9). Our observation that OCTN2 has the ability to transport carnitine in a sodium-dependent manner prompted us to search for mutations in the gene encoding OCTN2, SLC22A5. Initially, we analysed the mouse gene and found a missense mutation in Slc22a5 in jvs mice. Biochemical analysis revealed that this mutation abrogates carnitine transport. Subsequent analysis of the human gene identified four mutations in three SCD pedigrees. Affected individuals in one family were homozygous for the deletion of a 113-bp region containing the start codon. In the second pedigree, the affected individual was shown to be a compound heterozygote for two mutations that cause a frameshift and a premature stop codon, respectively. In an affected individual belonging to a third family, we found a homozygous splice-site mutation also resulting in a premature stop codon. These mutations provide the first evidence that loss of OCTN2 function causes SCD.

Functional relevance of carnitine transporter OCTN2 to brain distribution of l‐carnitine and acetyl‐l‐carnitine across the blood–brain barrier

Transport of l‐[3H]carnitine and acetyl‐l‐[3H]carnitine at the blood–brain barrier (BBB) was examined by using in vivo and in vitro models. In vivo brain uptake of acetyl‐l‐[3H]carnitine, determined by a rat brain perfusion technique, was decreased in the presence of unlabeled acetyl‐l‐carnitine and in the absence of sodium ions. Similar transport properties for l‐[3H]carnitine and/or acetyl‐l‐[3H]carnitine were observed in primary cultured brain capillary endothelial cells (BCECs) of rat, mouse, human, porcine and bovine, and immortalized rat BCECs, RBEC1. Uptakes of l‐[3H]carnitine and acetyl‐l‐[3H]carnitine by RBEC1 were sodium ion‐dependent, saturable with Km values of 33.1 ± 11.4 µm and 31.3 ± 11.6 µm, respectively, and inhibited by carnitine analogs. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was confirmed to be expressed in rat and human BCECs by an RT‐PCR method. Furthermore, the uptake of acetyl‐l‐[3H]carnitine by the BCECs of juvenile visceral steatosis (jvs) mouse, in which OCTN2 is functionally defective owing to a genetical missense mutation of one amino acid residue, was reduced. The brain distributions of l‐[3H]carnitine and acetyl‐l‐[3H]carnitine in jvs mice were slightly lower than those of wild‐type mice at 4 h after intravenous administration. These results suggest that OCTN2 is involved in transport of l‐carnitine and acetyl‐l‐carnitine from the circulating blood to the brain across the BBB.

Primary defect of juvenile visceral steatosis (jvs) mouse with systemic carnitine deficiency is probably in renal carnitine transport system

We investigated the reabsorptional system for carnitine in the kidney to elucidate the mechanism of carnitine deficiency in juvenile visceral steatosis (jvs) mice. Jvs mice had a higher rate of carnitine excretion at 10 days after birth than the controls, in spite of having no pathological acylcarnitine excretion in the urine. In an experiment to assay the uptake of carnitine using kidney slices, homozygous mutants showed significantly lower rates of Na-dependent carnitine uptake than controls. Heterozygous mice showed values of transport activity intermediate between homozygous mutants and homozygous controls. Scatchard plots (transport activity versus transport activity/carnitine concentration) revealed that the homozygous mutants had a defect in the high affinity site (Km = 58 microM) in the Na-dependent carnitine transport system in the kidney. These results indicate that the primary defect of jvs mice is most probably related to the system for reabsorption of carnitine in the kidney.

Cardiac hypertrophy in juvenile visceral steatosis (jvs) mice with systemic carnitine deficiency.

We have reported the clinical and biochemical findings in juvenile visceral steatosis (jvs) mice with systemic carnitine deficiency. This paper is the first report about cardiomyopathy in jvs mice. Adult jvs mice (at the age of 2 3 months) show cardiac hypertrophy which is caused by enlargement of the cardiac muscle cell associated with increases of non‐collagen protein and DNA content. Carnitine administration (2 mg/head, twice a day, from 1 month of age) significantly suppresses the cardiac hypertrophy, showing that carnitine deficiency plays an important role in the development of the cardiac hypertrophy. The discovery of cardiac hypertrophy in carnitine‐deficient jvs mice will lead to clarification of the pathophysiology of cardiomyopathy in systemic carnitine deficiency in human beings.

Gene-Dose Effect on Carnitine Transport Activity in Embryonic Fibroblasts of JVS Mice as a Model of Human Carnitine Transporter Deficiency

Recently, the marked decline in renal carnitine reabsorption has been thought to account fotr the systemic carnitine deficiency in juvenile visceral steatosis (JVS) mice. We have conducted a kinetic analysis using embryonic fibroblasts derived from normal, heterozygous, and homozygous jvs mice and found that the high-affinity carnitine transporter (Km = 5.5 microM), which shows Na+ and temperature dependency and stereospecificity, is defective in homozygous jvs mice. Moreover, a gene dose-dependent decrease of carnitine transport activity, which was due to a decrease in the number of the transporter molecules, was found in heterozygous jvs mice. Similar phenomena have been observed in human primary carnitine deficiency. Therefore, JVS mice may be useful for understanding this extremely rare human hereditary disorder.

Abnormal expression of urea cycle enzyme genes in juvenile visceral steatosis (jvs) mice

Juvenile visceral steatosis (jvs) mice from the C3H-H-2 degrees strain have markedly low levels of all the hepatic urea cycle enzymes (Imamura et al. (1990) FEBS Lett. 260, 119-121). The steady state levels of messenger RNA for the four urea cycle enzymes examined and also for albumin and serine dehydratase were severely reduced in the liver. The levels of mRNA for other liver-specific enzymes including aldolase B and phospho enol pyruvate carboxykinase did not vary significantly from normal littermates. As for extrahepatic expression of the urea cycle enzymes, only argininosuccinate synthetase in the kidney was decreased. Nuclear run-on experiments showed reduced transcription of the corresponding genes, which mostly accounts for the low mRNA levels. Furthermore, the time-course of mRNA accumulation from 5 days of age showed that the developmental induction of hepatic carbamyl phosphate synthetase and argininosuccinate synthetase mRNAs was strongly suppressed. These results suggest that jvs affects not only the regulation of the tissue-specific expression of the urea cycle enzymes but also the regulation of their developmental induction.

Mitochondrial abnormalities of muscle tissue in mice with juvenile visceral steatosis associated with systemic carnitine deficiency.

A mouse with juvenile visceral steatosis (the JVS mouse) has been recognized as a novel animal model for systemic carnitine deficiency. We examined cardiac, skeletal and smooth muscle cells in JVS and control mice by light and electron microscopy. Cardiac and skeletal muscle cells of these mice at 4 weeks of age exhibited a ragged-red appearance after trichrome staining. Electron microscopy, demonstrated increased numbers of mitochondria and lipid droplets in the cells. Compression or distortion of the myofibril bundles, primarily due to the increased number of mitochondria, suggests the possible existence of a functional disturbance of the cardiac and skeletal muscle. In the urinary bladder, only one or two large lipid droplets and slightly increased number of mitochondria were recognized in the perinuclear region of the smooth muscle cells. At 8 weeks of age, the mouse enzyme histochemistry specific for mitochondria, such as cytochrome c oxidase and succinic dehydrogenase, and oil red O staining, confirmed further increases in the number of mitochondria and lipid droplets in the heart. However, the accumulation of these organelles in the skeletal and smooth muscle cells was no greater than that noted in JVS mice at 4 weeks of age. In the cardiac muscle cells, autolysosomes or autophagic vacuoles containing electron-dense membranous, lamellar or whorled structures closely associated with mitochondria and pseudoinclusion bodies in the nucleus were recognized, and bundles of myofibrils were buried under numerous mitochondria, suggesting the existence of disturbed contractile function in the heart of JVS mice. These results indicate that this murine strain associated with systemic carnitine deficiency exhibits a generalized mitochondrial abnormality in the muscle system especially in the heart.

Disordered expression of glycolytic and gluconeogenic liver enzymes of juvenile visceral steatosis mice with systemic carnitine deficiency.

A quantitative study of the effect of carnitine deficiency on expression of glycolytic and gluconeogenic enzymes was performed using juvenile visceral steatosis mice which are systemically deficient in carnitine. The amounts of glucokinase and L-type pyruvate kinase mRNA were reduced in homozygotes, compared to heterozygotes and normal controls at 2 and 8 weeks. Liver-type phosphofructokinase, however, did not differ significantly. The abundance of fructose 1,6-bisphosphatase mRNA was unchanged at 2 and 8 weeks. The level of phosphoenolpyruvate carboxykinase mRNA was increased slightly at 2 weeks, but not at 8 weeks. A part of these changes could not be explained by the plasma glucose or insulin level. Carnitine administration restored the mRNA of these enzymes to normal levels. These results suggest that carnitine deficiency affects the expression of these liver enzymes.

Assignment of the gene locus for the sixth component (C6) of complement in mice to chromosome 15

A structural locus (C-6) for the sixth component of complement in mice is assigned to chromosome 15. Three-point linkage analysis indicated that the order of loci is C-6, Gpt-1, Gdc-1, and that the map distances are 25.9±4.9 between C-6 and Gpt-1, and 36.4±5.5 between C-6 and Gdc-1. Since Gdc-1 is more distal than Gpt-1, and C-6 is 26 cM away from Gpt-1, it is estimated that the C-6 is proximal to the centromere. In addition, a new C6 form found in AKR mice is described. We propose the designation C6B for it and C-6b for the allele encoding C6B.

Genetic polymorphism of the sixth component of complement (C6) in mice

Immunofixation after isoelectric focusing revealed two forms of mouse C6, C6A and C6M, both of which consist of two major protein bands and one or more acidic minor bands. They were distinguishable by their different isoelectric point (pI) ranges: C6M has more acidic pI ranges (pH < 6.2) than C6A (pH < 6.3). C6A was found in common inbred mice of Mus musculus domesticus, while C6M was found in inbred and wild mice of M. m. molossinus (Japanese wild mice, an Asian subspecies). Breeding experiments showed that these two forms of C6 were controlled by a single codominant autosomal locus. We propose the designation C-6 for this locus with two alleles, C-6aand C-6m, which encode for C6A and C6M, respectively. Linkage analysis indicated that the locus is not closely linked to the following loci: Idh-1, agouti, Amy-1, brown, Gpd-1, Mup-1, Pgm-2, Pgm-1, albino, Hbb, Es-1, Mod-1, Sep-1, Es-3, Igh-1, beige, Es-10, Sod-1, and C-3.