Miharu Ushikai

Identification of two novel mutations in the SLC25A13 gene and detection of seven mutations in 102 patients with adult-onset type II citrullinemia

Adult-onset type II citrullinemia (CTLN2) is characterized by a liver-specific deficiency of argininosuccinate synthetase (ASS) protein. We have recently identified the gene responsible for CTLN2, viz., SLC25A13, which encodes a calcium-binding mitochondrial carrier protein, designated citrin, and found five mutations of the SLC25A13 gene in CTLN2 patients. In the present study, we have identified two novel mutations, 1800ins1 and R605X, in SLC25A13 mRNA and the SLC25A13 gene. Diagnostic analysis for the seven mutations in 103 CTLN2 patients diagnosed by biochemical and enzymatic studies has revealed that 102 patients had one or two of the seven mutations and 93 patients were homozygotes or compound heterozygotes. These results indicate that CTLN2 is caused by an abnormality in the SLC25A13 gene, and that our criteria for CTLN2 before DNA diagnosis are correct. Five of 22 patients from consanguineous unions have been shown to be compound heterozygotes, suggesting a high frequency of the mutated genes. The frequency of homozygotes is calculated to be more than 1 in 20,000 from carrier detection (6 in 400 individuals tested) in the Japanese population. We have detected no cross-reactive immune materials in the liver of CTLN2 patients with any of the seven mutations by Western blot analysis with anti-human citrin antibody. From these findings, we hypothesize that CTLN2 is caused by a complete deletion of citrin, although the mechanism of ASS deficiency is still unknown.

Centrally administered nesfatin-1 inhibits feeding behaviour and gastroduodenal motility in mice.

Nesfatin-1 was recently identified as a peptide with anorexigenic effects that is localized in the hypothalamus and adipocytes. Not much is known about the effect of nesfatin-1 on gut motility. Food intake was measured after intracerebroventricular administration of nesfatin-1 in food-deprived mice. Antral and duodenal motility was assessed by using a manometric method in conscious fed mice. We found that centrally administered nesfatin-1 decreased food intake and inhibited gastroduodenal motility in mice. These results suggest that nesfatin-1 influences gut motility and feeding behaviour.

Identification of 13 novel mutations including a retrotransposal insertion in SLC25A13 gene and frequency of 30 mutations found in patients with citrin deficiency.

AbstractDeficiency of citrin, liver-type mitochondrial aspartate-glutamate carrier, is an autosomal recessive disorder caused by mutations of the SLC25A13 gene on chromosome 7q21.3 and has two phenotypes: neonatal intrahepatic cholestatic hepatitis (NICCD) and adult-onset type II citrullinemia (CTLN2). So far, we have described 19 SLC25A13 mutations. Here, we report 13 novel SLC25A13 mutations (one insertion, two deletion, three splice site, two nonsense, and five missense) in patients with citrin deficiency from Japan, Israel, UK, and Czech Republic. Only R360X was detected in both Japanese and Caucasian. IVS16ins3kb identified in a Japanese CTLN2 family seems to be a retrotransposal insertion, as the inserted sequence (2,667-nt) showed an antisense strand of processed complementary DNA (cDNA) from a gene on chromosome 6 (C6orf68), and the repetitive sequence (17-nt) derived from SLC25A13 was found at both ends of the insert. All together, 30 different mutations found in 334 Japanese, 47 Chinese, 11 Korean, four Vietnamese and seven non-East Asian families have been summarized. In Japan, IVS16ins3kb was relatively frequent in 22 families, in addition to known mutations IVS11 + 1G > A, 851del4, IVS13 + 1G > A, and S225X in 189, 173, 48 and 30 families, respectively; 851del4 and IVS16ins3kb were found in all East Asian patients tested, suggesting that these mutations may have occurred very early in some area of East Asia.

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

AbstractDeficiency 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.

Plasma nesfatin-1 concentrations in restricting-type anorexia nervosa

Restricting-type anorexia nervosa (AN-R) is characterized by chronic food restriction and severe emaciation due to various cognitive biases such as a distorted self-image. In spite of several treatments, AN-R continues to be a refractory disease because of its unknown pathogenesis. Although previous studies have shown that changes in feeding regulatory peptides such as ghrelin are involved in anorexia, few reports have described the relationship between AN-R and nesfatin-1, a recently identified satiety peptide. Therefore, we examined the plasma nesfatin-1 levels in AN-R patients to determine its role in AN-R. A total of 15 women participated in the study; 7 patients with AN-R and 8 age-matched healthy controls (average BMI, 13.02 ± 0.30 vs. 21.57 ± 0.48, respectively). Our results showed that plasma nesfatin-1 levels were significantly lower in AN-R group than in control group (6.23 ± 0.70 ng/ml vs. 8.91 ± 0.85 ng/ml, respectively, P<0.05). Plasma acyl ghrelin and des-acyl ghrelin levels were significantly higher in AN-R group than in control group (acyl ghrelin: 62.4 ± 10.15 fmol/ml vs. 27.20 ± 5.60 fmol/ml, P<0.01 and des-acyl ghrelin: 300.17 ± 55.95 fmol/ml vs. 107.34 ± 40.63 fmol/ml, P<0.05). Although AN-R is associated with emaciation for a prolonged period, our result suggested that nesfatin-1 levels may be regulated by nutrition status and response to starvation.

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.

Ghrelin reactive autoantibodies in restrictive anorexia nervosa

OBJECTIVE Subjects with restrictive anorexia nervosa (AN) display increased basal plasma levels of ghrelin that normalize after refeeding. The mechanism responsible for increased ghrelin levels in AN is unknown. We studied if changes of ghrelin reactive autoantibodies (autoAbs) could explain elevated plasma ghrelin in AN. METHODS Plasma levels of autoAbs reactive with ghrelin and des-acyl ghrelin were measured by enzyme-linked immunosorbent assay in subjects with AN before and 1 mo after hospitalization (refeeding) and compared with healthy controls and with plasma levels of ghrelin peptides. RESULTS Decreased levels of immunoglobulin (Ig) G, IgM, and IgA classes of autoAbs reacting with acyl ghrelin were found in patients with AN. Addition of des-acyl ghrelin but not of acyl ghrelin peptides at 10(-8) M to plasma before enzyme-linked immunosorbent assay showed in patients with AN but not in controls high levels of IgG autoAbs reacting with des-acyl ghrelin as a result of dissociation of des-acyl ghrelin autoAbs in immune complexes. Plasma levels of acyl and des-acyl ghrelin peptides correlated negatively with des-acyl ghrelin IgG autoAbs. Body mass index, which improved after refeeding, correlated with an increase of acyl ghrelin IgM autoAbs. CONCLUSION These results show that in patients with AN, ghrelin IgG autoAbs exist mainly as immune complexes with des-acyl ghrelin accompanied by a decrease of a free fraction of these autoAbs binding acylated and des-acyl ghrelin. This decrease of bioavailable ghrelin autoAbs may underlie a long-term elevation of plasma ghrelin levels and the resulting phenomenon of ghrelin resistance in malnourished patients with AN.

Comparison of the anorexigenic activity of CRF family peptides.

Corticotropin releasing factor (CRF) family peptides have an important role in the control of food intake. We investigated the effects of CRF family peptides on food intake and body weight gain in mice. Of the CRF family peptides, including CRF, urocortin1 (Ucn1), urocortin2 (Ucn2) and urocortin3 (Ucn3), peripherally administered Ucn1 was shown to have the most potent inhibitory effect on the food intake and body weight gain of both lean and high fat fed obese mice. In addition, repeated administration of Ucn1 lowered blood glucose and acylated ghrelin, and decreased the visceral fat weight of high fat fed obese mice.

Chronic foot-shock stress potentiates the influx of bone marrow-derived microglia into hippocampus

For several years, a new population of microglia derived from bone marrow has been described in multiple settings such as infection, trauma, and neurodegenerative disease. The aim of this study was to investigate the migration of bone marrow‐derived cells to the brain parenchyma after stress exposure. Stress exposure was performed in mice that had received bone marrow transplantation from GFP mice, allowing identification of blood‐derived elements within the brain. Electric foot‐shock exposure was chosen because of its ability to serve as fundamental and physical stress in mice. Bone marrow‐derived GFP+ cells migrated to the ventral part of the hippocampus and acquired a ramified microglia‐like morphology. Microglia marker Iba1 was expressed by 100% of the ramified cells, whereas ramified cells were negative for the astrocyte marker GFAP. Compared with the case in the control group, ramified cells significantly increased after chronic exposure to stress (5 days). One month after 5 days of stress exposure, ramified cells significantly decreased in ventral hippocampus compared with the group examined immediately after the last stress exposure. We report for the first time the migration of bone marrow‐derived cells to the ventral hippocampus after stress exposure. These cells have the characteristics of microglia. Mechanisms responsible for this migration and their roles in the brain remain to be determined.

Localization of il‐1β mrna and cell adhesion molecules in the maxillary sinus mucosa of patients with chronic sinusitis

Interleukin‐1β (IL‐1β) is a predominant cytokine in retained paranasal sinus fluid of chronic sinusitis where infiltration by polymorphonuclear neutrophils (PMNs) of nasal and paranasal mucosa is characteristic. The authors investigated the localization of IL‐1β messenger RNA (mRNA) in the maxillary sinus mucosa of patients with chronic sinusitis, using digoxigenin‐labeled oligonucleotide probes. IL‐1β mRNA was detected in some extravascular PMNs and small numbers of mononuclear leukocytes but was not detected in other tissue cells or intravascular leukocytes. The expression and distribution of the cell adhesion molecules, intercellular adhesion molecule‐1 (ICAM‐1) and endothelial‐leukocyte adhesion molecule‐1 (ELAM‐1), were also studied in cultured human mucosal microvascular endothelial cells and in the maxillary sinus mucosa in chronic sinusitis by immunohistochemistry using monoclonal antibodies against these cell adhesion molecules. Only ICAM‐1 was expressed on cultured human mucosal microvascular endothelial cells without IL‐1β stimulation. With IL‐1β activation of these cells, ELAM‐1 was expressed strongly and the expression of ICAM‐1 was enhanced. In the maxillary sinus mucosa, ICAM‐1 was strongly and universally expressed on endothelial cells of all small vessels, whereas ELAM‐1 was expressed only in the subepithelial region. These findings suggest that IL‐1β, one of mediators in chronic sinusitis, is produced by PMNs, induces the expression of ICAM‐1 and ELAM‐1 on endothelial cells, and, thereby, stimulates PMN infiltration in chronic sinusitis.