Juvenile Visceral Steatosis Mice Research Articles

Impact of alpha-tocopherol on cardiac hypertrophy due to energy metabolism disorder: the involvement of 1,2-diacylglycerol.

The juvenile visceral steatosis (JVS) mouse, a murine model of systemic carnitine deficiency, shows a disorder of fatty acid oxidation and develops cardiac hypertrophy associated with lipid accumulation. Recently, alpha-tocopherol was shown to decrease 1,2-diacylglycerol (DAG) levels. We investigated the involvement of DAG in cardiac hypertrophy due to energy metabolism disorder by evaluating the effects of alpha-tocopherol administration on the hearts of JVS mice. Both JVS and control mice were fed a high alpha-tocopherol diet or a standard diet from 4 to 8 weeks of age. Myocardial DAG levels and fatty acid composition were assessed at 8 weeks of age. The ventricular to body weight ratio in the JVS mice was significantly higher than that in the control mice [11.2+/-0.1 (mean+/-S.E.M.) versus 3.8+/-0.1 mg/g, P<0.01], and was reduced by alpha-tocopherol treatment (9.7+/-0.2 mg/g, P<0.01 versus JVS mice). However, echocardiographic analysis showed the exaggeration of left ventricular dilatation in the alpha-tocopherol treated JVS mice (P<0.01 versus JVS mice). The myocardial thiobarbituric-acid-reactive substance level was not affected by alpha-tocopherol treatment. The myocardial DAG level was 2.5-fold higher in the JVS mice compared with that in the control mice (2004+/-136 versus 806+/-36 ng/mg dry weight, P<0.01) with a significant increase in 18:1 and 18:2 fatty acids. alpha-Tocopherol treatment reduced myocardial DAG levels in the JVS mice (1443+/-49 ng/mg dry weight, P<0.01 versus JVS mice) without any alteration of the fatty acid composition. alpha-Tocopherol treatment may partially reduce cardiac hypertrophy but it may also depress cardiac function in the JVS mice by decreasing the myocardial DAG level. An increase in DAG might be involved in the development of cardiac hypertrophy and in the maintenance of cardiac function in energy metabolism disorder of the heart.

Biopharmaceutical Studies on Molecular Mechanisms of Membrane Transport

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Carnitine transport: pathophysiology and metabolism of known molecular defects.

Early-onset dilatative and/or hypertrophic cardiomyopathy with episodic hypoglycaemic coma and very low serum and tissue concentrations of carnitine should alert the clinician to the probability of the plasmalemmal high-affinity carnitine transporter defect. The diagnosis can be established by demonstration of impaired carnitine uptake in cultured skin fibroblasts or lymphoblasts and confirmed by mutation analysis of the human OCTN2 gene in the affected child and obligate heterozygote parents. The institution of high-dose oral carnitine supplementation reverses the pathology in this otherwise lethal autosomal recessive disease of childhood, and carnitine therapy from birth in prospectively screened siblings may altogether prevent the development of the clinical phenotype. Heterozygotes may be at risk for cardiomyopathy in later adult life, particularly in the presence of additional risk factors such as hypertension and competitive pharmacological agents. OCTN2 belongs to a family of organic cation/carnitine transporters that function primarily in the elimination of cationic drugs and other xenobiotics in kidney, intestine, liver and placenta. The high- and low-affinity human carnitine transporters, OCTN2 and OCTN1, are multifunctional polyspecific organic cation transporters; therefore, defects in these transporters may have widespread implications for the absorption and/or elimination of a number of key pharmacological agents such as cephalosporins, verapamil, quinidine and valproic acid. A third organic/cation carnitine transporter with high specificity for carnitine, Octn3, has been cloned in mice. The juvenile visceral steatosis (jvs) mouse serves as an excellent clinical, biochemical and molecular model for the high-affinity carnitine transporter OCTN2 defect and is due to a spontaneous point mutation in the murine Octn2 gene on mouse chromosome 11, which is syntenic to the human locus at 5q31 that harbours the human OCTN2 gene.

Changes in distinct species of 1,2-diacylglycerol in cardiac hypertrophy due to energy metabolic disorder.

The juvenile visceral steatosis (JVS) mouse, a genetic model of systemic carnitine deficiency resulting from carnitine transport mutation, develops cardiac hypertrophy. We determined two putative lipid messengers, 1,2-diacylglycerol (DAG) and ceramide, in JVS and carnitine palmitoyltransferase-I (CPT-I) inhibitor etomoxir-treated mice because these lipids function as co-messengers in the myocardium via modification of protein kinase C activity. JVS mice were evaluated at 4 and 8 weeks of age. The effect of long-term etomoxir treatment (45 mg/day) (ET) on mice was investigated in control mice from 4 to 8 weeks of age. As a model of inhibited cardiac hypertrophy, carnitine-treated JVS (CT) mice were produced. Myocardial DAG and ceramide levels and their fatty acid composition were measured. The heart/body weight ratio increased by 100% in JVS mice compared with that in controls, while that of CT mice was normalized in comparison with controls at 8 weeks of age. DAG markedly increased in both JVS and ET mice compared with that in controls (1,677+/-84, 1,258+/-49, and 585+/-58 ng/dry wt, respectively; P<0.01 for controls versus JVS or ET mice), whereas it was decreased significantly in CT mice compared with that in JVS mice (1,066+/-54 ng/dry wt, P<0.01). Furthermore, the fatty acid composition of DAG was similar in JVS and ET mice; in particular, 18:1 and 18:2 were significantly elevated in the myocardium (P<0.01 versus controls). On the other hand, that of DAG in CT mice was similar to that of the control group. In contrast, no difference was observed in myocardial ceramide levels among the groups. Pharmacological intervention with etomoxir mimics changes in the lipid second messenger characteristic of genetic JVS mice. The results suggest that the increases in distinct DAG species might be involved in the pathogenesis of cardiac hypertrophy as a result of disorder of fatty acid transport.

Functional Disorders of the Oxidative Phosphorylation System in the Heart Mitochondria of Mice with Juvenile Visceral Steatosis.

Mice with juvenile visceral steatosis (JVS) develop remarkable cardiac hypertrophy and exhibit an increased number of mitochondria in their heart. However, the biochemical characteristics and physiological functions of these mitochondria cardiac are little known. Here we show that the respiratory activities at state 3 with glutamate plus malate or succinate in the heart mitochondria of JVS mice were greatly decreased to 47% or 77%, respectively, compared with those of control mice. The contents of cytochromes a+a(3), b, and c+c(1) in the heart mitochondria of these mice were also decreased, to 51%, 45%, and 79%, respectively, of those of the control mice. Oligomycin-sensitive ATPase activitiy in these mitochondria, however, was increased to about 2 times over that of the control mice. Surprisingly, the ATP-Pi exchange activity of the heart mitochondria of JVS mice was greatly decreased, to 35% of that of control mice. On the other hand, the expression levels of 2 subunits of H(+)-ATP synthase, i.e., coupling factor 6 and alpha subunit, in heart mitochondria from control and JVS mice were almost the same. These results indicate that the coordinate regulation of mitochondrial proliferation and gene expression for components of the oxidative phosphorylation system was markedly defective in the heart of JVS mice. Our current results also suggest the presence of a novel regulatory mechanisms of ATP synthase activities in the heart.

Biopharmaceutical studies on molecular mechanisms of membrane transport

By incorporating the transporter-mediated or receptor-mediated transport process in physiologically based pharmacokinetic models, we succeeded in the quantitative prediction of plasma and tissue concentrations of beta-lactam antibiotics, insulin, pentazocine, quinolone antibacterial agents, and inaperizone and digoxin. The author's research on transporter-mediated pharmacokinetics focuses on the molecular and functional characteristics of drug transporters such as oligopeptide transporter, monocarboxylic acid transporter, anion antiporter, organic anion transporters, organic cation/carnitine transporters (OCTNs), and the ATP-binding cassette transporters P-glycoprotein and MRP2. We have successfully demonstrated that these transporters play important roles in the influxes and/or effluxes of drugs in intestinal and renal epithelial cells, hepatocytes, and brain capillary endothelial cells that form the blood-brain barrier. In the systemic carnitine deficiency (SCD) phenotype mouse model, juvenile visceral steatosis (jvs) mouse, a mutation in the OCTN2 gene was found. Furthermore, several types of mutation in human SCD patients were found, demonstrating that OCTN2 is a physiologically important carnitine transporter. Interestingly, OCTNs transport carnitine in a sodium-dependent manner and various cationic drugs transport it in a sodium-independent manner. OCTNs are thought to be multifunctional transporters for the uptake of carnitine into tissue cells and for the elimination of intracellular organic cationic drugs.

Novel mRNA molecules are induced in hypertrophied ventricles of carnitine-deficient mice and belong to a family of up-regulated gene in cells overexpressing c-erbB-2

To clarify the pathogenesis of cardiac hypertrophy in carnitine-deficient juvenile visceral steatosis (JVS) mice, we performed differential mRNA display analysis with the ventricles of control and JVS mice. We found a novel up-regulated gene, designated as carnitine deficiency-associated gene expressed in ventricle (CDV)-3. Northern blot analysis with a cDNA probe derived from the novel gene revealed two substantial mRNA species of prominent 4.1- and faint 3.5-kb in examined tissues of control and JVS mice. In spite of their widely expressed features, up-regulation of the gene was found predominantly in the ventricles and slightly in the auricles and skeletal muscles of JVS mice. The up-regulation of CDV-3 gene in the ventricles of JVS mice was significantly relieved by carnitine administration within 6 h. The entire cDNA nucleotide sequences showed that two kinds of cDNA, long and short versions (CDV-3A and -3B), corresponding to the detected mRNAs, are different in a 711 base fragment. Analysis of genomic DNA revealed that the two mRNAs were derived from a single CDV-3 gene with five exons by alternative splicing. The deduced amino acid sequences indicated that the isoforms consist of 236 and 281 residues, differing at regions near the carboxy-terminus but sharing 231 residues of the amino-terminal regions. A BLAST search revealed that they show a high similarity to a human predicted nuclear protein (H41), which has been reported to be up-regulated in breast cancer cells overexpressing cellular-erythroblastosis B-2 (c-erbB-2, a kind of tyrosine kinase). We report the identification and characterization of novel transcripts that may be involved in the development of cardiac hypertrophy caused by carnitine deficiency.

Age-associated cardiomyopathy in heterozygous carrier mice of a pathological mutation of carnitine transporter gene, OCTN2.

The purpose of this study was to test whether heterozygotes of juvenile visceral steatosis mice, a model for systemic carnitine deficiency, may develop age-associated cardiomyopathy. Tissue morphological observations were carried out by light and electron microscopy to compare the heterozygous and age-matched control mice at periods of 1 and 2 years. Possible effects of the pathological mutation on lipid and glucose levels was also evaluated in humans and mice. Except mild increases in serum cholesterol levels in male heterozygous mice and humans, no changes were found in other factors, indicating that none of the confounding factors seems to be profound. Results demonstrated that heterozygous mice had larger left ventriclular myocyte diameters than the control mice. Morphological changes in cardiac muscles by electron microscopy revealed age-associated changes of lipid deposition and abnormal mitochondria in heterozygous mice. Two out of 60 heterozygous cohort and one out of nine heterozygous trim-kill mice had cardiac hypertrophy at ages older than 2 years. The present study and our previous work suggest that the carrier state of OCTN2 pathological mutations might be a risk factor for age-associated cardiomyopathy.

A heterozygote phenotype is present in the jvs +/− mutant mouse livers

The juvenile visceral steatosis ( jvs) mouse, having a mutation in the carnitine transporter gene Octn2, is a model of primary systemic carnitine deficiency in humans (SCD, OMIM 212140). Like humans with SCD, homozygous jvs −/− mice have hepatic and cardiac steatoses, reduced plasma and tissue carnitines, and increased urinary carnitine clearance. Because symptomatic heterozygotes have been reported for some fatty acid oxidation disorders, including SCD, we compared the jvs heterozygotes to normal control mice. We measured the free and esterified carnitine, total cholesterol, and triglycerides in adult liver samples, myocardium, and skeletal muscle. Our results indicate significant differences between the livers of nonfasting adult normal ( n=8) vs jvs heterozygotes ( n=8) ( means± SEM, p<0.01) for the following parameters: free carnitine, 2.28±0.36 nmol/ mg protein vs 0.41±0.13; total carnitine, 3.48±0.36 vs 1.27±0.25; triglycerides, 0.14±0.04 vs 0.39±0.02; and total cholesterol, 0.21±0.02 vs 0.39±0.04, but not for esterified carnitine, 1.18±0.17 vs 0.90±0.17 ( p>0.05). There is also a negative correlation between hepatic free carnitine and triglycerides from jvs heterozygotes ( p<0.05). Similar results were obtained with myocardium and skeletal muscle. We conclude that free and total carnitine levels are significantly lower in the heterozygote mouse liver and heart while triglyceride and total cholesterol levels are significantly higher. We speculate that in situations of lipolytic stress, some SCD heterozygotes might develop clinical symptoms of carnitine deficiency.

Hyperammonemia in carnitine-deficient adult JVS mice used by starvation.

Juvenile visceral steatosis (JVS) mouse is an animal model of human primary carnitine deficiency caused by a mutation of the gene encoding carnitine transporter, and suffers from various symptoms, such as fatty liver, growth retardation, hyperammonemia, hypoglycemia, and cardiac hypertrophy. We have shown that hyperammonemia during the weaning period (15-26 days of age) is caused by suppression of urea cycle enzyme gene expression. The suppression resulted from activation of a transcription factor, AP-1. We have found that a cis-element for AP-1 binding is present in the enhancer region of the carbamoylphosphate synthetase (CPS) gene, and that the AP-1 binding site is involved in the suppression of CPS induction by dexamethasone in cultured hepatocytes and in the suppression of CPS expression in the liver of JVS mice. The blood ammonia levels in JVS mice increased during the weaning period, and then decreased to almost control levels after 30 days of age. In this paper, we report that in adult JVS mice, ammonia levels again increased after starvation for at least 24 hr and this effect was suppressed by carnitine treatment. Starvation for 48 hr did not significantly suppress CPS activity in the liver and did not cause any change in hepatic ornithine concentration. The concentration of N-acetylglutamate in the liver of starved JVS mice was not significantly different from that of JVS mice treated with carnitine. These results indicate that the hyperammonemia in carnitine-deficient adult JVS mice during starvation and the suppression by carnitine treatment differ from those found during the weaning period, and thus the cause of hyperammonemia and the mechanism of suppression remain to be solved.

Activities of Cytochrome P450 Enzymes in Liver and Kidney Microsomes from Systemic Carnitine Deficiency Mice with a Gene Mutation of Carnitine/Organic Cation Transporter

Juvenile visceral steatosis (jvs) mice, isolated from the C3H-H-2 degrees strain, exibit a systemic carnitine deficiency (SCD) phenotype and develop fatty liver, hyperammonemia and hypoglycemia. This phenotype is caused by a missense mutation (Leu352Arg) of a sodium-dependent carnitine/organic cation transporter, Octn2 (Slc22a5). The jvs mouse could be a useful model for pharmacokinetics and drug metabolism studies concerning Octn2 substrate drugs. In the present study, the effects of the SCD phenotype on the cytochrome P450 (P450 or CYP) dependent activities of four endobiotic and seven xenobiotic oxidations catalyzed by liver and kidney microsomes from jvs mice were investigated. The jvs-type mutation was genotyped by PCR-RFLP. The contents of total P450 and NADPH-P450 reductase were similar in the the liver microsomes from male or female mice of the wild-type and those heterozygous or homozygous for the jvs-type mutation. The 6beta-hydroxylation activities of testosterone and progesterone (marker for Cyp3a) based on the protein contents were 1.2- to 2.0-fold higher in liver microsomes from jvs/jvs-type mice compared to jvs/wt- or wt/wt-type mice. Coumarin 7-hydroxylation activities (marker for Cyp2a) were decreased to 0.7-fold in the male jvs/jvs-type mice. The activities of lauric acid 12-hydroxylation (a marker for Cyp4a) and aniline p-hydroxylation (a marker for Cyp2e1) in liver microsomes were increased 1.4- to 1.9-fold in female jvs/jvs-type mice. Genotoxic activation of 2-aminofluorene (a marker for Cyp4b1) by male and female mouse kidney microsomes were not affected by the SCD phenotype. These results demonstrated that the SCD phenotype affected the P450-dependent catalytic activities in liver microsomes. The jvs mouse could provide valuable information in drug interaction and drug metabolism studies of OCTN2 substrate drugs and new compounds in development.

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 K(m) values of 33.1 +/- 11.4 microM and 31.3 +/- 11.6 microM, 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.

Antioxidant changes in the hypertrophied heart due to energy metabolic disorder

Oxidative stress has been implicated in the pathogenesis of both heart hypertrophy and heart failure. Hypertrophied heart, in response to pressure overload, is associated with an increase in antioxidant capacity and a decrease in oxidative stress. However, in the hypertrophied heart due to energy metabolic disorder, antioxidant capacity has not been investigated. Antioxidant changes in juvenile visceral steatosis (JVS) mice, a model of heart hypertrophy due to disorder of fatty-acid oxidation, were examined at 4 weeks (developing hypertrophy stage) and 8 weeks of age (established hypertrophy stage). Superoxide dismutase activity in the JVS mice was higher than that in control mice at 4 weeks of age and was not different from that in the control mice at 8 weeks of age. Glutathione peroxidase activity in the JVS mice at 8 weeks of age was lower than that in the control mice. Catalase activity showed no significant differences between the control and the JVS mice. Lipid peroxidation in the JVS mice was significantly reduced at 4 weeks of age and increased toward control levels at 8 weeks of age. The levels of vitamin E in the heart were increased in the JVS mice at 8 weeks of age. To determine whether antioxidants affect the pathogenesis of hypertrophy in this model, long-term treatments of vitamin E and 2-mercaptopropionyl glycine were performed. Vitamin E treatment partially reduced the heart hypertrophy in these mice. The present study shows that heart hypertrophy in the JVS mice is accompanied with increased antioxidant capacity as indicated in other animal models of heart hypertrophy. The precise mechanism of heart hypertrophy in JVS mice is still unknown, but oxidative stress may play a role in the pathogenesis of heart hypertrophy.

Carnitine Transport by Organic Cation Transporters and Systemic Carnitine Deficiency

The intracellular homeostasis is controlled by different membrane transporters. Organic cation transporters function primarily in the elimination of cationic drugs, endogenous amines, and other xenobiotics in tissues such as the kidney, intestine, and liver. Among these molecules, carnitine is an endogenous amine which is an essential cofactor for mitochondrial β-oxidation. Recently, a new family of transporters, named OCT (organic cation transporters) has been described. In this minireview, we present the recent knowledge about OCT and focus on carnitine transport, more particularly by the OCTN2. The importance of this sodium-dependent carnitine cotransporter, OCTN2, comes from various recently reported mutations in the gene which give rise to the primary systemic carnitine deficiency (SCD; OMIM 212140). The SCD is an autosomal recessive disorder of fatty acid oxidation characterized by skeletal myopathy, progressive cardiomyopathy, hypoglycemia and hyperammonemia. Most of the OCTN2 mutations identified in humans with SCD result in loss of carnitine transport function. Identifying these mutations will allow an easy targeting of the SCD syndrome. The characteristics of the juvenile visceral steatosis (jvs) mouse, an animal model of SCD showing similar symptoms as humans having this genetic disorder, are also described. These mice have a mutation in the gene encoding the mouse carnitine transporter octn2. Although various OCTN carnitine transporters have been identified and functionally characterized, their membrane localization and regulation are still unknown and must be investigated. This knowledge will also help in designing new drugs that regulate carnitine transport activity.

Involvement of 1,2-diacylglycerol in cardiac hypertrophy of the juvenile visceral steatosis mice with systemic carnitine deficiency

Involvement of 1,2-diacylglycerol in cardiac hypertrophy of the juvenile visceral steatosis mice with systemic carnitine deficiency

Effect of gamma-butyrobetaine on fatty liver in juvenile visceral steatosis mice.

We pharmacokinetically examined the effect of gamma-butyrobetaine, a precursor of L-carnitine, on the change of fatty acid metabolism in juvenile visceral steatosis (JVS) mice, which have systemic L-carnitine deficiency due to lack of L-carnitine transporter activity. The concentrations of total free fatty acid (FFA), palmitic acid and stearic acid in the liver of JVS mice were significantly higher than those in wild-type mice. After intravenous administration of gamma-butyrobetaine (50 mg kg(-1)), the concentration of L-carnitine in the plasma of JVS mice reached about twice that of the control level and levels in the brain, liver and kidney were also significantly increased, whereas those in wild-type mice hardly changed. Although the plasma concentrations of FFA in both types of mice were unchanged after administration of gamma-butyrobetaine, the concentrations of palmitic acid and stearic acid were significantly decreased. In particular, the liver concentration of FFA in JVS mice was decreased to the wild-type control level, accompanied by significant decreases in long-chain fatty acids, palmitic acid and stearic acid, whereas those in wild-type mice were not changed. These results suggest that gamma-butyrobetaine can be taken up into organs, including the liver, of JVS mice, and transformed to L-carnitine. Consequently, administration of gamma-butyrobetaine may be more useful than that of L-carnitine itself for treatment of primary deficiency of carnitine due to a functional defect of the carnitine transporter.

Molecular and physiological evidence for multifunctionality of carnitine/organic cation transporter OCTN2.

OCTN2 is an Na(+)-dependent transporter for carnitine, which is essential for fatty acid metabolism, and its functional defect leads to fatal systemic carnitine deficiency (SCD). It also transports the organic cation tetraethylammonium (TEA) in an Na(+)-independent manner. Here, we studied the multifunctionality of OCTN2, by examining the transport characteristics in cells transfected with mouse OCTN2 and in juvenile visceral steatosis (jvs) mice that exhibit a SCD phenotype owing to mutation of the OCTN2 gene. The physiological significance of OCTN2 as an organic cation transporter was confirmed by using jvs mice. The embryonic fibroblasts from jvs mice exhibited significantly decreased transport of [(14)C]TEA. Pharmacokinetic analysis of [(14)C]TEA disposition demonstrated that jvs mice showed decreased tissue distribution and renal secretory clearance. In transport experiments using OCTN2-expressing cells, TEA and carnitine showed mutual trans-stimulation effects in their transport, implying a carnitine/TEA exchange mechanism. In addition, Na(+) affected the affinity of carnitine for OCTN2, whereas Na(+) is unlikely to be involved in TEA transport. This is the first molecular and physiological demonstration of the operation of an organic cation transporter in renal apical membrane. The results are consistent with the physiological coupling of carnitine reabsorption with the secretion of organic cations.

Antagonizing Effect of AP-1 on Glucocorticoid Induction of Urea Cycle Enzymes: A Study of Hyperammonemia in Carnitine-Deficient, Juvenile Visceral Steatosis Mice

Hyperammonemia is one of the major symptoms of primary carnitine deficiency. Carnitine-deficient juvenile visceral steatosis (JVS) mice show hyperammonemia during the weaning period. We have found that all of the urea cycle enzyme genes are suppressed and that N-acetylglutamate, an allosteric activator of the first step enzyme of the urea cycle, carbamoyl phosphate synthetase I (CPS), is not deficient in the liver of JVS mice. Induction of the urea cycle enzymes by glucocorticoid in rat primary cultured hepatocytes was suppressed by the addition of long-chain fatty acids. The suppression of the urea cycle enzyme genes in vivo and in vitro is accompanied by stimulated AP-1 DNA-binding activity. However, mRNA of phosphoenolpyruvate carboxykinase, one of the gluconeogenic enzymes which responds to glucocorticoid, is further stimulated by the addition of fatty acid. From these results, we postulate that protein-protein interaction between glucocorticoid receptors and AP-1 is not the major mechanism of suppression, but that AP-1 causes the suppression through a cis-element on the gene. After cloning promoter and enhancer regions of the mouse CPS gene and comparing rat and mouse, we found that an AP-1 site was present just 3′-downstream of the minimal essential enhancer fragment previously described. We also found that the presence of an AP-1 site in reporter gene constructs resulted in suppression of the reporter genes in the liver of carnitine-deficient JVS mice and suppression of glucocorticoid induction by long-chain fatty acid in cultured hepatocytes.

Genomic organization and mapping of mouse CDV (carnitine deficiency-associated gene expressed in ventricle)-1 and its related CDV-1R gene.

We have previously reported that CDV (carnitine deficiency-associated gene expressed in ventricle)-1 was a downregulated gene in the hypertrophied ventricle of carnitine-deficient juvenile visceral steatosis mice and that the related gene (CDV-1R) showed no tissue specificity and no sensitivity to carnitine deficiency. In the present paper, the CDV-1/1R gene was isolated from a mouse genomic BAC library, and the genomic structure was characterized. We found that the CDV-1/1R gene consisted of at least 19 exons and encompassed approximately 48 kb. The splice sites conformed to the GT-AG rule, and the CDV-1R mRNA containing 19 exons was processed. CDV-1 mRNA containing 5 exons was constructed from the 3' half of CDV-1R. The first exon of CDV-1 consisted of the 3' side (116 bp) of intron 14 and exon 15 (87 bp) of CDV-1R. The presumed promoter sequence for CDV-1 located in the intron 14 of CDV-1R contained the common TATA box and consensus binding sites for various transcription factors (Nkx-2.5, Spl, C/EBP, SRF, YY1, and CREB), which seem to play roles in the heart-specific expression and carnitine deficiency-associated suppression of CDV-1. In the upstream region of the CDV-1 promoter, we found two VNTRs, 13 repeats of GATA1, and 16 copies of STRE involved in yeast stress response. The CDV-1/1R gene was located close to DSMIT68 on mouse Chromosome (Chr) 5, corresponding to human Chr 12q24. All these data revealed that two mRNA species, CDV-1 and CDV-1R, are expressed tissue-specifically by using promoters peculiar to each transcript in a single gene.

Involvement of a cis-Acting Element in the Suppression of Carbamoyl Phosphate Synthetase I Gene Expression in the Liver of Carnitine-Deficient Mice

The expression of carbamoyl phosphate synthetase I (CPS) gene is suppressed in the liver of carnitine-deficient juvenile visceral steatosis (JVS) mice at weaning and under starvation at adult age. To clarify the suppression mechanism, we produced CPSL transgenic JVS mice carrying a transgene composed of the chloramphenicol acetyltransferase (CAT) gene with the upstream region (−12 kb to +138) of the rat CPS gene and CPSE transgenic JVS mice carrying a transgene composed of the luciferase gene with minimal promoter (299 bp from −161 to +138) and enhancer (469 bp around −6.3 kb) fragments of the rat gene. The expression of the CAT gene as well as the endogenous CPS was suppressed in CPSL transgenic JVS mice, but luciferase gene expression was not suppressed in CPSE transgenic JVS mice. We isolated the 5′-upstream region of the mouse CPS gene and identified an activator protein-1 (AP-1) site downstream of the minimum enhancer region of both rat and mouse CPS genes. In conjunction with the 313-bp mouse promoter region, the 714-bp mouse enhancer fragment conferred a cell-type-dependent hormone responsiveness. In rat primary cultured hepatocytes, the addition of oleic acid suppressed reporter gene expression induced by dexamethasone in the construct containing the enhancer fragment of 714 bp with the AP-1 site, but not in its AP-1 site mutants or in 519 bp without the AP-1 site. These results strongly suggest that direct protein–protein interaction between AP-1 and glucocorticoid receptor is not involved in the suppression of the CPS gene in JVS mice and that the AP-1 element is the cis-element which is responsible for the suppression.