Increased insulin sensitivity despite lipodystrophy in Crebbp heterozygous mice.

The CBP protein (cAMP response element binding protein (CREB) binding protein) is a co-activator for several transcription factors with a wide range of important biological functions, such as sterol regulatory element binding proteins (SREBPs), CCAAT/enhancer-binding proteins (C/EBPs), nuclear receptors (including peroxisome proliferator-activated receptors, PPARs), and signal transducers and activators of transcription (STATs). In contrast to these individual transcription factors, the biological roles of CBP are poorly understood. CBP enhances transcriptional activities via histone acetylation and the recruitment of additional co-activators such as SRC (steroid coactivator)-1 (ref. 9). To identify its physiological functions using a loss-of-function mutant, we analyzed CBP-deficient mice. As Crebbp null mice (Crebbp-/-) died during embryogenesis, we used Crebbp+/- mice. Unexpectedly, Crebbp+/- mice showed markedly reduced weight of white adipose tissue (WAT) but not of other tissues. Despite this lipodystrophy, Crebbp+/- mice showed increased insulin sensitivity and glucose tolerance and were completely protected from body weight gain induced by a high-fat (HF) diet. We observed increased leptin sensitivity and increased serum adiponectin levels in Crebbp+/- mice. These increased effects of insulin-sensitizing hormones secreted from WAT may explain, at least in part, the phenotypes of Crebbp+/- mice. This study demonstrates that CBP may function as a 'master-switch' between energy storage and expenditure.

Exploring the conformational dynamics and thermodynamics of EGFR S768I and G719X + S768I mutations in non-small cell lung cancer: An in silico approaches.

Non-small cell lung cancer (NSCLC) is often driven by mutations in the epidermal growth factor receptor (EGFR) gene. However, rare mutations such as G719X and S768I lack standard anti-EGFR targeted therapies. Understanding the structural differences between wild-type EGFR and these rare mutants is crucial for developing EGFR-targeted drugs. We performed a systematic analysis using molecular dynamics simulations, essential dynamics (ED), molecular mechanics Poisson-Boltzmann surface area, and free energy calculation methods to compare the kinetic properties, molecular motion, and free energy distribution between wild-type EGFR and the rare mutants' structures G719X-EGFR, S768I-EGFR, and G719X + S768I-EGFR. Our results showed that S768I-EGFR and G719X + S768I-EGFR have higher global and local conformational flexibility and lower thermal and global structural stability than WT-EGFR. ED analysis revealed different molecular motion patterns between S768I-EGFR, G719X + S768I-EGFR, and WT-EGFR. The A-loop and αC-helix, crucial structural elements related to the active state, showed a tendency toward active state development, providing a molecular mechanism explanation for NSCLC caused by EGFR S768I and EGFR G719C + S768I mutations. The present study may be helpful in the development of new EGFR-targeted drugs based on the structure of rare mutations. Our findings may aid in developing new targeted treatments for patients with EGFR S768I and EGFR G719X + S768I mutations.

Prolonged effect of single carnitine administration on fasted carnitine-deficient JVS mice regarding their locomotor activity and energy expenditure.

Carnitine is an essential cofactor for the oxidation of fatty acid in the mitochondria and an efficient therapeutics for primary carnitine deficiency. We herein analyzed the prolonged effects of carnitine on the reduced locomotor activity and energy metabolism of fasted carnitine-deficient juvenile visceral steatosis (jvs(-/-)) mice. We found that a single carnitine administration to 24-h fasted jvs(-/-) mice in the morning increased both the locomotor activity and oxygen consumption at night not only on the same day, but also on the next day, when the carnitine levels in the blood and tissues were already as low as at the original carnitine-deficient state. We also found that fat utilization for energy production significantly increased under fasting even in jvs(-/-) mice and was stimulated in the carnitine-administrated fasted jvs(-/-) mice at night, in comparison to that observed in the saline-administered jvs(-/-) mice, at least for 2 days even under the low plasma and tissue carnitine levels. These results suggest that the low tissue carnitine levels are therefore not the sole rate-limiting factor of general fatty acid oxidation in carnitine-deficient jvs(-/-) mice.

Slc25a13-knockout mice harbor metabolic deficits but fail to display hallmarks of adult-onset type II citrullinemia.

Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resulting Ctrn-/- mice were devoid of Slc25a13 mRNA and citrin protein. Liver mitochondrial assays revealed markedly decreased activities in aspartate transport and the malate-aspartate shuttle. Liver perfusion also demonstrated deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate-to-pyruvate ratio within hepatocytes. Surprisingly, Ctrn-/- mice up to 1 year of age failed to show CTLN2-like symptoms due to normal hepatic ASS activity. Serological measures of glucose, amino acid, and ammonia metabolism also showed no significant alterations. Nitrogen-loading treatments produced only minor changes in the hepatic ammonia and amino acid levels. These results suggest that citrin deficiency alone may not be sufficient to produce a CTLN2-like phenotype in mice. These observations are compatible, however, with the variable age of onset, incomplete penetrance, and strong ethnic bias seen in CTLN2 where additional environmental and/or genetic triggers are now suspected.

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.

Fasting-induced reduction in locomotor activity and reduced response of orexin neurons in carnitine-deficient mice.

We found reduced locomotor activity (LA) under fasting in systemic carnitine-deficient juvenile visceral steatosis (jvs(-/-)) mice. When food was withdrawn at 8:00 a.m. (lights-off at 7:00 p.m., 12h/cycle), the nocturnal LA of jvs(-/-) mice was much less than the control (jvs(+/+) and jvs(+/-)) mice. LA recovered under carnitine or sucrose administration, but not under medium-chain triglyceride. In addition, fasted jvs(-/-) mice, without any energy supply, were activated by modafinil, a stimulator of the dopamine pathway. These results suggest that the reduced LA is not adequately explained by energy deficit. As the fasted jvs(-/-) mice showed lower body core temperature (BT), we examined the central nervous system regulating LA and BT. We found lower percentage of c-Fos positive orexin neurons in the lateral hypothalamus and reduced orexin-A concentration in the cerebrospinal fluid of fasted jvs(-/-) mice. Sleep analysis revealed that fasted jvs(-/-) mice had disruption of prolonged wakefulness, with a higher frequency of brief episodes of non-REM sleep during the dark period than fasted jvs(+/+) mice. These results strongly suggest that the reduced LA in fasted jvs(-/-) mice is related to the inhibition of orexin neuronal activity.

[Progresses and perspectives in the study on citrin deficiency].

Citrin deficiency causes autosomal recessive disorders including adult-onset type II citrullinemia (CTLN2) and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). The responsive gene of citrin deficiency, SLC25A13, locates on chromosome 7q21.3 and encodes citrin as a liver-type mitochondrial aspartate/glutamate carrier (AGC). The mutations on SLC25A13 will result in deficiency of citrin and CTLN2 or NICCD. Citrin deficiency was found at first in Japan. However, recently, some of cases were identified in China, Korea, Vietnam, Israel, Czech, United States and England, and racial differences of the SLC25A13 mutations were found, suggesting the patients with citrin deficiency maybe exist worldwide. In this article, authors reviewed the progresses in the study on citrin deficiency up to now and put forward authors' considerations for further research on it.

Expression of three mitochondrial solute carriers, citrin, aralar1 and ornithine transporter, in relation to urea cycle in mice.

The present report describes the expression profiles of different tissues and developmental changes of mouse aspartate/glutamate carrier (AGC) genes, Slc25a13 and Slc25a12, and an ornithine transporter gene, Ornt1, in relation to urea cycle enzyme genes, carbamoylphosphate synthetase I (CPS) and argininosuccinate synthetase (ASS). Slc25a13 encodes citrin, recently found to be deficient in adult-onset type II citrullinemia and to function as AGC together with its isoform and product of Slc25a12, aralar1. Citrin was broadly distributed, but mainly in the liver, kidney and heart. Aralar1 was expressed in diaphragm, skeletal muscle, heart, brain and kidney, but not in the liver. These distribution profiles are different from the restricted of Ornt1, ASS and CPS. Citrin, ASS, CPS and Ornt1 showed similar patterns of developmental changes in the liver and small intestine, where they play a role in urea and arginine synthesis. Dietary, hormonal and physical manipulations caused varied changes of CPS, ASS and Ornt1 in the liver, but the change of citrin was not so marked as that of the others. Analysis using RT-PCR and restriction enzyme digestion revealed that the ornithine transporter most expressed is Ornt1, although Ornt2 is detectable at a minute level. All these results suggest that citrin as AGC plays a role in urea synthesis as well as many fundamental metabolic pathways in the liver, and shares metabolic functions with aralar1 in other tissues, and that Ornt1 is an important component in urea synthesis in the liver and in arginine synthesis in the small intestine during the neonatal period.

Citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice recapitulate features of human citrin deficiency.

Citrin is the liver-type mitochondrial aspartate-glutamate carrier that participates in urea, protein, and nucleotide biosynthetic pathways by supplying aspartate from mitochondria to the cytosol. Citrin also plays a role in transporting cytosolic NADH reducing equivalents into mitochondria as a component of the malate-aspartate shuttle. In humans, loss-of-function mutations in the SLC25A13 gene encoding citrin cause both adult-onset type II citrullinemia and neonatal intrahepatic cholestasis, collectively referred to as human citrin deficiency. Citrin knock-out mice fail to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity may be compensating for the loss of citrin function in the mouse, we have generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase. The resulting double knock-out mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and a fatty liver, all features of human citrin deficiency. An increased hepatic lactate/pyruvate ratio in the double knock-out mice compared with controls was also further elevated by the oral sucrose administration, suggesting that an altered cytosolic NADH/NAD(+) ratio is closely associated with the hyperammonemia observed. Microarray analyses identified over 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, revealing genes potentially involved in compensatory or downstream effects of the combined mutations. Together, our data indicate that the more severe phenotype present in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice represents a more accurate model of human citrin deficiency than citrin knock-out mice.

Pyruvate ameliorates the defect in ureogenesis from ammonia in citrin-deficient mice.

BACKGROUND/AIMS: Mutations in SLC25A13, encoding the mitochondrial aspartate-glutamate carrier citrin, cause adult-onset type II citrullinemia (CTLN2) in humans. We have previously reported that although citrin-knockout (Ctrn-/-) mice fail to display symptoms of CTLN2, liver perfusion revealed a deficit in ureogenesis from ammonia accompanied by an increase in the perfusate lactate-to-pyruvate (L/P) ratio. The present study explores the effects of pyruvate, aspartate and citrate on improving the abnormalities observed in the Ctrn-/- liver. METHODS: We measured the rate of ureogenesis from ammonium chloride using the liver-perfusion system. RESULTS: Pyruvate infusion lowered the L/P ratio and corrected the deficit in ureogenesis in the Ctrn-/- liver. This effect was found to be dose-dependent in both instances. Phenazine methosulfate, a cytosolic oxidant, also improved the rate of ureogenesis in the Ctrn-/- liver and led to a fall in the L/P ratio. The addition of aspartate or citrate did not change either the rate of ureogenesis or the L/P ratio in the Ctrn-/- liver. CONCLUSIONS: Citrin deficiency disturbs urea synthesis primarily as a result of an elevated cytosolic NADH/NAD+ ratio owing to limited reoxidation of reducing equivalents. Clinically, pyruvate may have a therapeutic benefit for CTLN2 patients.

Frequency and distribution in East Asia of 12 mutations identified in the SLC25A13 gene of Japanese patients with citrin deficiency.

Deficiency of citrin, a liver-type mitochondrial aspartate-glutamate carrier (AGC), encoded by the SLC25A13 gene on chromosome 7q21.3, causes autosomal recessive disorders: adult-onset type II citrullinemia (CTLN2) and neonatal hepatitis associated with intrahepatic cholestasis (NICCD). So far, we have described 12 SLC25A13 mutations: 11 were from Japan and one from Israel. Three mutations found in Chinese and Vietnamese patients were the same as those in Japanese patients. In the present study, we identified a novel mutation IVS6+1G>C in a Japanese CTLN2 patient and widely screened 12 SLC25A13 mutations found in Japanese patients in control individuals from East Asia to confirm our preliminary results that the carrier frequency was high in Asian populations. Mutations 851-854del and 1638-1660dup were found in all Asian countries tested, and 851-854del associated with 290-haplotype in microsatellite marker D7S1812 was especially frequent. Other mutations frequently detected were IVS11+1G>A in Japanese and Korean, S225X in Japanese, and IVS6+5G>A in Chinese populations. We found a remarkable difference in carrier rates in China (including Taiwan) between north (1/940) and south (1/48) of the Yangtze River. We detected many carriers in Chinese (64/4169 = 1/65), Japanese (20/1372 = 1/69) and Korean (22/2455 = 1/112) populations, suggesting that over 80,000 East Asians are homozygotes with two mutated SLC25A13 alleles.

Reduced N-acetylaspartate levels in mice lacking aralar, a brain- and muscle-type mitochondrial aspartate-glutamate carrier.

Aralar is a mitochondrial calcium-regulated aspartate-glutamate carrier mainly distributed in brain and skeletal muscle, involved in the transport of aspartate from mitochondria to cytosol, and in the transfer of cytosolic reducing equivalents into mitochondria as a member of the malate-aspartate NADH shuttle. In the present study, we describe the characteristics of aralar-deficient (Aralar-/-) mice, generated by a gene-trap method, showing no aralar mRNA and protein, and no detectable malate-aspartate shuttle activity in skeletal muscle and brain mitochondria. Aralar-/- mice were growth-retarded, exhibited generalized tremoring, and had pronounced motor coordination defects along with an impaired myelination in the central nervous system. Analysis of lipid components showed a marked decrease in the myelin lipid galactosyl cerebroside. The content of the myelin lipid precursor, N-acetylaspartate, and that of aspartate are drastically decreased in the brain of Aralar-/- mice. The defect in N-acetylaspartate production was also observed in cell extracts from primary neuronal cultures derived from Aralar-/- mouse embryos. These results show that aralar plays an important role in myelin formation by providing aspartate for the synthesis of N-acetylaspartate in neuronal cells.

Pathogenesis and pathophysiology of citrin (a mitochondrial aspartate glutamate carrier) deficiency.

Adult-onset type II citrullinemia (CTLN2), characterized by a liver-specific deficiency of urea cycle enzyme, argininosuccinate synthetase, is caused by mutations in SLC25A13 that encodes a calcium binding mitochondrial solute carrier protein, citrin. Citrin deficiency causes not only CTLN2 but also neonatal intrahepatic cholestasis caused by citrin deficiency at neonatal period. Moreover citrin and its isoform aralar were found to be aspartate glutamate carrier. From the viewpoint of the metabolic functions of citrin as aspartate glutamate carrier in urea synthesis and NADH shuttle, symptoms of CTLN2 and neonatal intrahepatic cholestasis caused by citrin deficiency are analyzed.

Adult-onset type II citrullinemia and idiopathic neonatal hepatitis caused by citrin deficiency: involvement of the aspartate glutamate carrier for urea synthesis and maintenance of the urea cycle.

Citrin is a mitochondrial aspartate glutamate carrier primarily expressed in the liver, heart, and kidney. We found that adult-onset type II citrullinemia is caused by mutations in the SLC25A13 gene that encodes for citrin. In this report, we describe the frequency of SLC25A13 mutations, the roles of citrin as a member of the urea cycle and as a member of the malate-aspartate shuttle, the relationship between its functions and symptoms of citrin deficiency, and therapeutic issues.