Carnitine-deficient Juvenile Visceral Steatosis Mice Research Articles

Failure of the feeding response to fasting in carnitine-deficient juvenile visceral steatosis (JVS) mice: Involvement of defective acyl-ghrelin secretion and enhanced corticotropin-releasing factor signaling in the hypothalamus

Carnitine-deficient juvenile visceral steatosis (JVS) mice, suffering from fatty acid metabolism abnormalities, have reduced locomotor activity after fasting. We examined whether JVS mice exhibit specific defect in the feeding response to fasting, a key process of anti-famine homeostatic mechanism. Carnitine-deficient JVS mice showed grossly defective feeding response to 24 h-fasting, with almost no food intake in the first 4 h, in marked contrast to control animals. JVS mice also showed defective acyl-ghrelin response to fasting, less suppressed leptin, and seemingly normal corticotropin-releasing factor (CRF) expression in the hypothalamus despite markedly increased plasma corticosterone. The anorectic response was ameliorated by intraperitoneal administration of carnitine or acyl-ghrelin, with decreased CRF expression. Intracerebroventricular treatment of CRF type 2 receptor antagonist, anti-sauvagine-30, recovered the defective feeding response of 24 h-fasted JVS mice. The defective feeding response to fasting in carnitine-deficient JVS mice is due to the defective acyl-ghrelin and enhanced CRF signaling in the hypothalamus through fatty acid metabolism abnormalities. In this animal model, carnitine normalizes the feeding response through an inhibition of CRF.

Attenuation of Cardiac Hypertrophy in Carnitine-Deficient Juvenile Visceral Steatosis (JVS) Mice Achieved by Lowering Dietary Lipid

We examined the development of cardiac hypertrophy in juvenile visceral steatosis (JVS) mice, a model of systemic carnitine deficiency, by varying the amount of lipid in the diet. Cardiac hypertrophy was markedly attenuated by decreasing soy bean oil (SBO) from 5% (w/w) to 1%. Triglyceride contents of the ventricles of JVS mice fed 1% SBO were significantly lower than in JVS mice fed 5% SBO. The addition of medium-chain triglycerides metabolically utilized by JVS mice did not affect the development of cardiac hypertrophy. On the other hand, the mRNA levels of atrial natriuretic peptide and skeletal alpha-actin, which are related to cardiac hypertrophy, were also attenuated by decreasing lipid in the diet. Adenylate energy charge and creatine phosphate in the heart of JVS mice at the early stage of hypertrophy were not significantly different from control mice given the same laboratory chow (4.6% of lipid). Although urinary prostaglandin F(2alpha) levels were found to be increased in JVS mice at 15 days of age when they developed cardiac hypertrophy, administration of aspirin was not efficacious. We, therefore, propose that the proportion of lipid in the diet is important in the development of cardiac hypertrophy in carnitine-deficient JVS mice, and that this is not related to prostaglandin formation.

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.

A novel gene suppressed in the ventricle of carnitine-deficient juvenile visceral steatosis mice

In order to clarify the pathogenesis and pathophysiology of cardiac hypertrophy in carnitine-deficient juvenile visceral steatosis (JVS) mice, we performed mRNA differential display analysis with total RNA extracted from the ventricles of control and JVS mice at 14 days of age. We identified four up-regulated genes, two known and two unknown, and a novel down-regulated gene. Northern blot analysis with a novel cDNA probe derived from the down-regulated gene fragment 8A2 revealed three mRNA species of 1.1-, 1.3-, and 2.6-kb. The 1.1- and 1.3-kb mRNA species were found only in the heart, and the 2.6-kb species was found in the heart, kidney and brain, but not in skeletal muscle or liver. The 1.1- and 1.3-kb species were down-regulated in the ventricles of JVS mice, but not in the auricles, and increased to the control level with carnitine treatment. We isolated cDNA clones from ventricle RNA, termed CDV-1 (carnitine deficiency-associated gene expressed in ventricle) and from brain RNA, termed CDV-1R (CDV-1-related gene) by 5′- and 3′-RACE analyses. The entire nucleotide sequence except the 5′-terminal 64 bp of CDV-1 cDNA was completely identical to the 992 bp sequence from the 3′-end of CDV-1R cDNA. The CDV-1 cDNA contained an open reading frame predicting a peptide of 107 amino acids, which composed the C-terminal portion of CDV-1R peptide consisting of 414 amino acids.

Increased expression of carnitine palmitoyltransferase I gene is repressed by administering L-carnitine in the hearts of carnitine-deficient juvenile visceral steatosis mice.

The juvenile visceral steatosis (JVS) mouse is a novel mutant animal for studying systemic carnitine deficiency. The importance of the model has been pointed out in carnitine-deficient cardiac hypertrophy, since cardiomyopathy has been often improved after oral carnitine therapy in human systemic carnitine deficiency. To understand the effects of carnitine deficiency on gene expression in the heart, we tried to find the genes regulated by carnitine by means of a modified differential display procedure. Carnitine palmitoyltransferase I (CPT I) was one of the isolated genes. The level of CPT I gene expression in the ventricles of the JVS mice was at least three- to sixfold that of normal mice as judged by reverse transcription-polymerase chain reaction (RT-PCR). When the JVS mice were treated with carnitine, CPT I gene expression was repressed to the level of normal mice. Therefore, the increased expression of the CPT I gene was associated with carnitine deficiency.