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Background/aim The effects of dietary protein and carbohydrate content on the plasma amino acid profile of patients with diabetes are not fully understood. Therefore, we examined whether there are effects of diets with differing proportions of protein and carbohydrate on the plasma amino acid concentrations of control (CT) mice and mice with type 2 diabetes (db). Materials and methods We used db mice as an animal model of type 2 diabetes which are genetically deficient in leptin receptor. Diets with differing proportions of protein and carbohydrates (L diet: low protein/carbohydrate ratio, H diet: high protein/carbohydrate ratio) were supplied. db Mice were fed with a restriction on the basis of the consumption by CT-L mice, such that equivalent amounts of energy and fat were consumed. In CT mice fed the L or H diets, there was no significant difference in ad libitum food intake. Results There were significant interactions between diet and genotype with respect to water intake, urine volume, urinary glucose concentration, and plasma isoleucine, leucine, valine, branched-chain amino acids, and serine concentrations. db-H mice showed significantly higher water intake, urine volume, and urinary glucose than db-L mice. db Mice fed the L or H diets had similar plasma amino acid profiles, except for valine. In contrast, CT-H mice showed significantly higher valine and branched-chain amino acids and lower serine concentrations than CT-L mice. Thus, the CT-H mice were more similar to db mice fed either of the diets. Conclusion There were different effects of the dietary protein or carbohydrate content on the plasma amino acid profiles between nondiabetic and diabetic mice. In particular, the profiles in nondiabetic conditions were different between the low- and high-protein diet conditions.

Previous studies using citrin/mitochondrial glycerol-3-phosphate (G3P) dehydrogenase (mGPD) double-knockout mice have demonstrated that increased dietary protein reduces the extent of carbohydrate-induced hyperammonemia observed in these mice. This study aimed to further elucidate the mechanisms of this effect. Specific amino acids were initially found to decrease hepatic G3P, or increase aspartate or citrulline levels, in mGPD-knockout mice administered ethanol. Unexpectedly, oral glycine increased ammonia in addition to lowering G3P and increasing citrulline. Subsequently, simultaneous glycine-plus-sucrose (Gly + Suc) administration led to a more severe hyperammonemic state in double-KO mice compared to sucrose alone. Oral arginine, ornithine, aspartate, alanine, glutamate and medium-chain triglycerides all lowered blood ammonia following Gly + Suc administration, with combinations of ornithine-plus-aspartate (Orn + Asp) or ornithine-plus-alanine (Orn + Ala) suppressing levels similar to wild-type. Liver perfusion and portal vein-arterial amino acid differences suggest that oral aspartate, similar to alanine, likely activated ureagenesis from ammonia and lowered the cytosolic NADH/NAD+ ratio through conversion to alanine in the small intestine. In conclusion, Gly + Suc administration induces a more severe hyperammonemic state in double-KO mice that Orn + Asp or Orn + Ala both effectively suppress. Aspartate-to-alanine conversion in the small intestine allows for effective oral administration of either, demonstrating a pivotal role of inter-organ aspartate metabolism for the treatment of citrin deficiency.

Background: Propofol inhibits fatty acid oxidation and induces mitochondrial deficiency, a possible mechanism involved in propofol infusion syndrome. This study investigated how propofol influences fatty acid, glucose, and amino acid metabolism, as well as whether L-carnitine may improve suppression of free fatty acid metabolism. Methods: Male Sprague-Dawley rats, fasted for 16 hours, were allocated to the following two groups: (Group P; continuous intravenous administration of 10 mg/kg/h propofol; n = 8) and (Group P + C; intravenous administration of 50 mg/kg and then 50 mg/kg/h L-carnitine continuously; n = 8). Concentrations of glucose, free fatty acid (FFA), amino acids, in-sulin, and β-hydroxybutyric acid were measured at the start and then one, two, and three hours after propofol administration. Intrahepatic triglyceride levels were measured at the end of experiments. In vitro experiments comprised measurement of oxygen consumption in human hepatocytes (Hepg2) and investigating dependency on palmitic acid, glucose, and glutamine as fuel during propofol administration, with or without L-carnitine. Results: FFA increased in Group P and gradually decreased in Group P + C. There were significant differences between the two groups (Group P; 331.2 ± 64.5 μM vs. Group P + C; 199 ± 73.6 μM). Glucose decreased in both groups (Group P; 53.8 ±16.6 mg/dL vs. Group P + C; 88 ± 11.3 mg/dL). Amino acid concentrations were higher in Group P + C after experiments; alanine and glutamine increased significantly. β-hydroxybutyric acid increased significantly in Group P + C, and intrahepatic triglyceride decreased in Group P + C. Dependency on fatty acid metabolism significantly decreased with propofol only; addition of L-carnitine prevented these effects. Conclusions: Propofol impaired mitochondrial fatty acid metabolism, which was compensated mainly by a switch to glucose metabolism and partially by amino acid metabolism. Addition of L-carnitine may improve this imbalance of energy metabolism.

Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients.

Helicobacter pylori (Hp) infection is related to the pathogenesis of chronic gastric disorders and extragastric diseases. Here, we examined the anorexigenic and anxiogenic effects of Hp vacuolating cytotoxin A (VacA) through activation of hypothalamic urocortin1 (Ucn1). VacA was detected in the hypothalamus after peripheral administration and increased Ucn1 mRNA expression and c-Fos-positive cells in the hypothalamus but not in the nucleus tractus solitarius. c-Fos and Ucn1-double positive cells were detected. CRF1 and CRF2 receptor antagonists suppressed VacA-induced anxiety and anorexia, respectively. VacA activated single paraventricular nucleus neurons and A7r5 cells; this activation was inhibited by phospholipase C (PLC) and protein kinase C (PKC) inhibitors. VacA causes anorexia and anxiety through the intracellular PLC-PKC pathway, migrates across the blood-brain barrier, and activates the Ucn1-CRF receptor axis.

Background/aim The aim of this study was to develop a method for sequentially collecting cerebrospinal fluid (CSF) from an unanesthetized microminipig, which shares many physiological and anatomical similarities with humans, such as diurnality, and investigate the diurnal variation of melatonin concentration in the CSF. Materials and methods A catheter was placed percutaneously into the subarachnoid space of an anesthetized animal, and the tip of the catheter was placed into the cisterna magna under X-ray. We then sequentially collected CSF at light-on and -off times from the unanesthetized animal for several weeks. After catheter placement, a period of one week or more was necessary to relieve the contamination of RBCs in the CSF. Results A higher melatonin level in the CSF was noted during lights-off time, and the level was higher than that in the serum. Conclusion This model of sequential collection of CSF will contribute to research in brain functions.

Background/aim Diabetic nephropathy is aggravated by a higher intake of total protein. The effects of diets with different proportions of protein and carbohydrate on diabetic retinopathy in db mice, a type-2 diabetes animal model, were examined, as well as diabetic nephropathy. Materials and methods Control and db mice at 5 weeks of age were fed the diets (% energy of protein/carbohydrate/fat; L-diet: 12/71/17; H-diet: 24/59/17) under ad libitum conditions and pair-feeding conditions for 6 weeks, respectively. Results Mice fed the H-diet showed significantly greater retinal thickness by optical coherence tomography, and lower mRNA levels of angiotensinogen. Comparing combinations of diets and genotypes, db-H mice showed significantly higher mRNA levels of angiotensin-converting enzyme, advanced glycosylation end product-specific receptor, and cluster of differentiation molecule 11b (a microglial marker) than db-L mice. Conclusion Dietary protein and carbohydrate proportions influenced retinal manifestations, including retinal thickness and gene expression in control and diabetic mice.

The effects of dietary and lighting conditions on diurnal rhythm of locomotor activity (LA) and body temperature (BT) using four adult male microminipigs were investigated. Different feeding times, diet and lighting conditions were applied sequentially for 3 weeks in each phase as follows: Phase I: Morning mealtime, normal diet, 12-h lights on; phase II: mealtime changed to afternoon; phase III: diet changed to high-fat diet; phase IV: lighting changed to 20-h on; and phase V: phase I repeated. LA was measured by an actigraph which was worn on the body of each pig. A BT recording module (Thermochron Type-SL) was implanted in the neck subcutaneously. Phase II increased BT compared with phase I. Phase III increased LA and BT compared with phase II. Phase IV increased LA compared with phase III. LA in phase V was higher compared with phase I. These results can be extrapolated to other diurnal animals such as humans. This study provides an example of the effects of diet and lighting on biological activities in microminipigs under low-invasive procedures measuring LA and BT, leading to low variations in these measures.

Different involvement of leptin signaling in food intake (FI) and body temperature (BT) in pups and adults has been suggested. However, the leptin receptor (Lepr) long-form-deficient (db) mouse line has not been fully examined in pups. In the most availabledbmouse line, wild-type (WT) mice have a mutation in the dedicator of cytokinesis 7 gene, namedmisty, which was recently revealed to be involved in neuronal development. Therefore, we established a line ofdbmice without themistymutation using natural mating. Adult (8 weeks of age) homozygousdb/dbmice displayed significantly higher core body weight (BW) and FI and significantly lower core BT than WT mice. However, postnatal (2 weeks of age)db/dbmice displayed similar BW and milk intake and significantly lower core BT than WT mice. Correspondingly, adult and postnataldb/dbmice exhibited altered mRNA levels of hypothalamic orexigenic and anorexigenic peptide in adults but not in pups. Additionally,db/dbmice displayed significantly lower mRNA levels of brown adipose tissue uncoupling protein 1 at both ages. In conclusion, thedbmouse line without themistymutation clearly showed the different involvements of the Lepr long form in FI and BT in pups and adults.

We previously showed the deleterious effects of increased dietary protein on renal manifestations and glucose metabolism in leptin receptor-deficient (db) mice. Here, we further examined its effects on glucose metabolism, including urinary C-peptide. We also orally administered mixtures corresponding to low- or high-protein diets to diabetic mice. In diet experiments, under pair-feeding (equivalent energy and fat) conditions using a metabolic cage, mice were fed diets with different protein content (L diet: 12 % protein, 71 % carbohydrate, 17 % fat; H diet: 24 % protein, 59 % carbohydrate, 17 % fat) for 15 days. In oral administration experiments, the respective mixtures (L mixture: 12 % proline, 71 % maltose or starch, 17 % linoleic acid; H mixture: 24 % proline, 59 % maltose or starch, 17 % linoleic acid) were supplied to mice. Biochemical parameters related to glucose metabolism were measured. The db–H diet mice showed significantly higher water intake, urinary volume, and glucose levels than db–L diet mice but similar levels of excreted urinary C-peptide. In contrast, control-H diet mice showed significantly higher C-peptide excretion than control-L diet mice. Both types of mice fed H diet excreted high levels of urinary albumin. When maltose mixtures were administered, db–L mixture mice showed significantly higher blood glucose after 30 min than db–H mixture mice. However, db mice administered starch–H mixture showed significantly higher blood glucose 120–300 min post-administration than db–L mixture mice, although both groups exhibited similar insulin levels. High-protein, low-carbohydrate diets deteriorated diabetic conditions and were associated with insufficient insulin secretion in db mice. Our findings may have implications for dietary management of diabetic symptoms in human patients.

The mitochondrial aspartate-glutamate carrier isoform 2 (citrin) and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse has been a useful model of human citrin deficiency. One of the most prominent findings has been markedly increased hepatic glycerol 3-phosphate (G3P) following oral administration of a sucrose solution. We aimed to investigate whether this change is detectable outside of the liver, and to explore the mechanism underlying the increased hepatic G3P in these mice. We measured G3P and its metabolite glycerol in plasma and urine of the mice under various conditions. Glycerol synthesis from fructose was also studied using the liver perfusion system. The citrin/mGPD double-knockout mice showed increased urine G3P and glycerol under normal, fed conditions. We also found increased plasma glycerol under fasted conditions, while oral administration of different carbohydrates or ethanol led to substantially increased plasma glycerol. Fructose infusion to the perfused liver of the double-knockout mice augmented hepatic glycerol synthesis, and was accompanied by a concomitant increase in the lactate/pyruvate (L/P) ratio. Co-infusion of either pyruvate or phenazine methosulfate, a cytosolic oxidant, with fructose corrected the high L/P ratio, leading to reduced glycerol synthesis. Overall, these findings suggest that hepatic glycerol synthesis is cytosolic NADH/NAD+ ratio-dependent and reveal a likely regulatory mechanism for hepatic glycerol synthesis following a high carbohydrate load in citrin-deficient patients. Therefore, urine G3P and glycerol may represent potential diagnostic markers for human citrin deficiency.

The food habit is involved in the onset and development of lifestyle-related diseases. In this review I would like to describe a historical case of vitamin B1 deficiency, as well as our case study of fatty acid metabolism abnormality due to carnitine deficiency. In history, the army and navy personnel in Japan at the end of the 19th century received food rations based on a high-carbohydrate diet including white rice, resulting in the onset of beriberi. An epidemiological study by Kenkan Takaki revealed the relationship between the onset of beriberi and rice intake. Then, Takaki was successful in preventing the onset of beriberi by changing the diet. However, the primary cause had yet to be elucidated. Finally, Christian Eijkman established an animal model of beriberi (chickens) showing peripheral neuropathy, and he identified the existence of an anti-beriberi substance, vitamin B1. This is an example of the successful control of a disease by integrating the results of epidemiological and experimental studies. In our study using a murine model of fatty acid metabolism abnormality caused by carnitine deficiency, cardiac abnormality and fatty liver developed depending on the amount of dietary fat. In addition, the mice showed disturbance of orexin neuron activity related to the sleep-arousal system, which is involved in fatigue symptoms under fasting condition, one of the states showing enhanced fatty acid metabolism. These findings suggest that fatty acid toxicity is enhanced when the mice are more dependent on fatty acid metabolism. Almost simultaneously, a human epidemiological study showed that narcolepsy, which is caused by orexin system abnormality, is associated with the polymorphism of the gene coding for carnitine palmitoyltransferase 1B, which is involved in carnitine metabolism. To understand the pathological mechanism of fatty acid toxicity, not only an experimental approach using animal models, but also an epidemiological approach is necessary. The results will be applied to preventing and treating lifestyle-related diseases associated with fatty acid metabolism abnormality.

Exercise is effective for preventing the onset and development of type 2 diabetes mellitus (T2DM) in human cases; however, the effect of exercise on the pathophysiology using animal models of T2DM has not been fully evaluated.We applied voluntary exercise under pair-fed (P) conditions in db mice, an animal model of T2DM. Exercising (Ex) and sedentary (Se) mice were placed in a cage, equipped with a free or locked running wheel, for 4 weeks, respectively. The amount of food consumed by ad libitum-fed wild-type mice under the Se condition (ad-WT) was supplied to all mice, except ad libitum db mice (ad-db). Blood parameters and expression of the genes involved in nutrient metabolism were analyzed.PEx-db (pair-fed and exercising) mice showed significantly lower HbA1c, body weight and liver weight than PSe-db and ad-db mice. Decreased hepatic triglycerides in PEx-db mice corresponded to a lower expression of lipogenic enzyme genes in the liver. Moreover, PEx-db mice showed significantly lower plasma branched-chain amino acids (BCAA), arginine, proline, and tyrosine, in addition to increased skeletal muscle (SM) weight, than PSe-db and ad-db mice, in spite of little influence on the expression of the BCAA transaminase gene, in SM and WAT.We found that exercise under a food restriction condition decreases several amino acids, including BCAA, and may improve insulin sensitivity more than mere food restriction. We propose that the decreased concentration of blood amino acids may be a valuable marker evaluating the effects of exercise on diabetic conditions.

Gastric and adipose tissue secrete a number of hormones that are involved in energy metabolism. The biological functions of these hormones, including their effects on aging, are currently under investigation. Adiponectin was shown to be directly involved in appetite and the control of body weight. However, the effects of aging of nesfatin-1, an appetite-suppressing peptide that was recently identified, have not yet been fully elucidated. The aim of this study was to determine the effects of aging on the plasma levels of nesfatin-1 and adiponectin. Our results demonstrated no significant differences in the nesfatin-1 plasma levels among three age groups (2, 6 and 24 months) of female BALB/c mice. The plasma nesfatin-1 levels/visceral fat (VF) ratio in the 24-month-old mice was significantly lower compared to that in the 2- and 6-month-old mice. In addition, there were no significant differences in the plasma adiponectin levels among the three age groups. The plasma adiponectin levels/VF ratio in the 24-month-old mice was significantly lower compared to that in the 2- and 6-month-old mice. In conclusion, there were no age-related changes in the plasma levels of nesfatin-1 and adiponectin, although the ratio of plasma levels of nesfatin-1 and adiponectin per VF was decreased with advancing age. Our results indicated that nesfatin-1 and adiponectin may be involved in controlling energy balance during aging.

The C57BL/6:Slc23a13(-/-);Gpd2(-/-) double-knockout (a.k.a., citrin/mitochondrial glycerol 3-phosphate dehydrogenase double knockout or Ctrn/mGPD-KO) mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2), making it a suitable model of human citrin deficiency. In the present study, we show that when mature Ctrn/mGPD-KO mice are switched from a standard chow diet (CE-2) to a purified maintenance diet (AIN-93M), this resulted in a significant loss of body weight as a result of reduced food intake compared to littermate mGPD-KO mice. However, supplementation of the purified maintenance diet with additional protein (from 14% to 22%; and concomitant reduction or corn starch), or with specific supplementation with alanine, sodium glutamate, sodium pyruvate or medium-chain triglycerides (MCT), led to increased food intake and body weight gain near or back to that on chow diet. No such effect was observed when supplementing the diet with other sources of fat that contain long-chain fatty acids. Furthermore, when these supplements were added to a sucrose solution administered enterally to the mice, which has been shown previously to lead to elevated blood ammonia as well as altered hepatic metabolite levels in Ctrn/mGPP-KO mice, this led to metabolic correction. The elevated hepatic glycerol 3-phosphate and citrulline levels after sucrose administration were suppressed by the administration of sodium pyruvate, alanine, sodium glutamate and MCT, although the effect of MCT was relatively small. Low hepatic citrate and increased lysine levels were only found to be corrected by sodium pyruvate, while alanine and sodium glutamate both corrected hepatic glutamate and aspartate levels. Overall, these results suggest that dietary factors including increased protein content, supplementation of specific amino acids like alanine and sodium glutamate, as well as sodium pyruvate and MCT all show beneficial effects on citrin deficiency by increasing the carbohydrate tolerance of Ctrn/mGPD-KO mice, as observed through increased food intake and maintenance of body weight.

We aimed to characterize the effects of preptin on insulin secretion at the single-cell level, as well as the mechanisms underlying these changes, with respect to regulation by intracellular Ca2+[Ca2+]imobilization. This study assessed the effect of preptin on insulin secretion and investigated the link between preptin and the phospholipase C (PLC)/protein kinase C (PKC) pathway at the cellular level using fura-2 pentakis(acetoxymethyl) ester-loaded insulin-producing cells (Min 6 cells). Our results demonstrate that preptin promotes insulin secretion in a concentration-dependent manner. Using a PLC inhibitor (chelerythrine) or a PKC inhibitor (U73122) resulted in a concentration-dependent decrease in insulin secretion. Also, preptin mixed with IGF2 receptor (IGF2R) antibodies suppressed insulin secretion in a dose-dependent manner, which indicates that activation of IGF2R is mediated probably because preptin is a type of proIGF2. In addition, preptin stimulated insulin secretion to a similar level as did glibenclamide. The activation of PKC/PLC by preptin stimulation is highly relevant to the potential mechanisms for increase in insulin secretion. Our results provide new insight into the insulin secretion of preptin, a secreted proIGF2-derived peptide that can induce greater efficacy of signal transduction resulting from PLC and PKC activation through the IGF2R.

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.

Pyruvate dehydrogenase kinase 4 (PDK4) mRNA has been reported as an up-regulated gene in the heart and skeletal muscle of carnitine-deficient juvenile visceral steatosis (JVS) mice under fed conditions. PDK4 plays an important role in the inhibition of glucose oxidation via the phosphorylation of pyruvate dehydrogenase complex (PDC). This study evaluated the meaning of increased PDK4 mRNA in glucose metabolism by investigating PDK4 protein levels, PDC activity and glucose uptake by the heart and skeletal muscle of JVS mice. PDK4 protein levels in the heart and skeletal muscle of fed JVS mice were increased in accordance with mRNA levels, and protein was enriched in the mitochondria. PDK4 protein was co-fractionated with PDC in sucrose density gradient centrifugation, like PDK2 protein; however, the activities of the pyruvate dehydrogenase complex (PDC) active form in the heart and skeletal muscle of fed JVS mice were similar to those in fed control mice. Fed JVS mice showed significantly higher glucose uptake in the heart and similar uptake in the skeletal muscle compared with fed control mice. Thus, in carnitine deficiency under fed conditions, glucose was preferentially utilized in the heart as an energy source despite increased PDK4 protein levels in the mitochondria. The preferred glucose utilization may be involved in developing cardiac hypertrophy from carnitine deficiency in fatty acid oxidation abnormality.

Urocortin 3 (Ucn3) is recognized as a member of the corticotropin-releasing factor (CRF) family, which plays an important role in regulating food intake. We investigated the effects of centrally administered Ucn3 on food intake and gastric emptying in mice. Intracerebroventricular (ICV) administration of Ucn3 (0.1–1 nmol per mouse) decreased food intake in a dose-dependent manner. The inhibitory effect of Ucn3 on food intake was less potent than that of centrally administered CRF and Urocortin 1. ICV administration of Ucn3 (0.1–1 nmol per mouse) decreased the gastric emptying rate in a dose-dependent manner. Ucn3 decreased food intake in high-fat diet-fed obese mice as well as in lean mice. These results indicated that Ucn3 influences feeding behavior and gut motility, and may be a promising therapeutic target in the treatment of eating and functional gastrointestinal disorders.

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.

The objective of this study was to clarify the role of a novel agonist with high selectivity and affinity for Y4 receptors in the regulation of food intake. The Y4 receptor agonist BVD-74D was administered to C57BL/6J mice that were fed with a normal or high-fat diet, and to fatty liver Shionogi (FLS)-ob/ob mice; the food intake, water intake, and body weight gain were measured in these mice. In the mice fed with a normal diet, the cumulative food intake significantly decreased at 20 min and 1 h after the administration of 1 mg/kg of BVD-74D and at 1, 2, 4, 8, and 24 h after the administration of 10 mg/kg of BVD-74D. Moreover, the cumulative water intake and body weight gain significantly decreased in these mice. Among the mice that were fed with a high-fat diet, the cumulative food intake and water intake significantly decreased 1, 2, and 4 h after BVD-74D (10 mg/kg) administration. Furthermore, the cumulative food intake significantly decreased 2 and 4 h after BVD-74D (10 mg/kg) administration in the FLS-ob/ob mice. Thus, we propose that the novel Y4 receptor agonist BVD-74D has suppressive effects on food intake, water intake, and weight gain in normal mice fed with normal diets and on food intake in normal mice fed with high-fat diets and in FLS-ob/ob mice. These findings indicate that the Y4 receptor and its agonist would be promising targets for obesity.

Restricting type of anorexia nervosa (AN-R) is a serious disorder affecting adolescents and young adults and decreases quality of life over a long period. Successful weight restoration is an important prognostic factor for disease outcome; however, the underlying mechanism of refeeding resistance, a core psychopathology relevant to 'ambivalent' eating behaviors, remains unclear in this disorder. Ghrelin plays an important role in the regulation of growth hormone release, appetite, and energy metabolism. However, the early progress of these patients and changes in the levels of acyl ghrelin and des-acyl ghrelin during treatment were not reported. The purpose of this study was to determine the changes in ghrelin levels (acyl and des-acyl) during early treatment. As a result, des-acyl ghrelin in AN-R patients is higher than in control subjects before the therapy, but it decreases with treatment. The plasma des-acyl ghrelin level in AN-R patients started decreasing more rapidly and in early stage of the hospitalization than ever reported, and after 8 weeks, it is significantly lower than in control subjects. It means that des-acyl ghrelin is sensitive and changeable with their nutrition state. Furthermore, the ratio of the acyl ghrelin to total ghrelin increases with 8 weeks treatment. Eight weeks after, energy intake of the AN-R patients is recovered near the normal range with a daily energy intake of 1 700+/-93.54 kcal. These findings may be valuable for future AN-R treatments in order to increase acyl ghrelin and decrease des-acyl ghrelin, thereby influencing the refeeding outcome.

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.

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.

Deficiency 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.

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.

Rice koji is considered a readily accessible functional food that may have health-promoting effects. We investigated whether white, yellow, and red koji have the anti-obesity effect in C57BL/6J mice fed a high-fat diet (HFD), which is a model for obesity. Mice were fed HFD containing 10% (w/w) of rice koji powder or steamed rice for 4 weeks. Weight gain, epididymal white adipose tissue, and total adipose tissue weight were significantly lower in all rice koji groups than in the HFD-rice group after 4 weeks. Feed efficiency was significantly reduced in the yellow koji group. Blood glucose levels were significantly lower in the white and red koji groups with HOMA-R and leptin levels being reduced in the white koji group. White and red koji increased glucose uptake and GLUT4 protein expression in L6 myotube cells. These results showed that all rice koji have the anti-obesity or anti-diabetes effects although the mechanisms may differ depending on the type of rice koji consumed.

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.

Hydrogen (H(2)) acts as a therapeutic antioxidant. However, there are few reports on H(2) function in other capacities in diabetes mellitus (DM). Therefore, in this study, we investigated the role of H(2) in glucose transport by studying cultured mouse C2C12 cells and human hepatoma Hep-G2 cells in vitro, in addition to three types of diabetic mice [Streptozotocin (STZ)-induced type 1 diabetic mice, high-fat diet-induced type 2 diabetic mice, and genetically diabetic db/db mice] in vivo. The results show that H(2) promoted 2-[(14)C]-deoxy-d-glucose (2-DG) uptake into C2C12 cells via the translocation of glucose transporter Glut4 through activation of phosphatidylinositol-3-OH kinase (PI3K), protein kinase C (PKC), and AMP-activated protein kinase (AMPK), although it did not stimulate the translocation of Glut2 in Hep G2 cells. H(2) significantly increased skeletal muscle membrane Glut4 expression and markedly improved glycemic control in STZ-induced type 1 diabetic mice after chronic intraperitoneal (i.p.) and oral (p.o.) administration. However, long-term p.o. administration of H(2) had least effect on the obese and non-insulin-dependent type 2 diabetes mouse models. Our study demonstrates that H(2) exerts metabolic effects similar to those of insulin and may be a novel therapeutic alternative to insulin in type 1 diabetes mellitus that can be administered orally.

Objective Anorexia nervosa (AN) continues to be a refractory disease because of its unknown pathogenesis. The role of adiponectin in AN has not been clarified. Moreover, few reports have described the relations between adiponectin isoforms and AN in the physical and psychological states. Therefore, we measured plasma adiponectin and its isoforms levels in patients with AN to examine their roles in AN. Methods Eighteen women participated in this study: nine patients with AN and nine age-matched healthy controls. We examined plasma adiponectin and its isoforms levels in all subjects and administered three types of psychological test to patients with AN: the Eating Disorders Inventory–2, the Maudsley Obsessional–Compulsive Inventory, and the Beck Depression Inventory–2. Results We found that the percentage of high-molecular-weight (HMW) to total adiponectin (%HMW) was significantly low and the percentage of low-molecular-weight (LMW) to total adiponectin (%LMW) was significantly high in the AN group compared with the control group. The %HMW positively and the %LMW negatively correlated with body mass index in the entire study population. The %HMW was also positively correlated with psychological symptoms such as social insecurity or cleaning evaluated with the Eating Disorders Inventory–2 or the Maudsley Obsessional–Compulsive Inventory. Conclusions Our study indicates that all adiponectin isoforms should be evaluated in patients with AN in addition to total adiponectin. The decreased %HMW and the increased %LMW that were correlated with the body mass index and some components of psychopathology in our patients may indicate a complex role of adiponectin isoforms in maintaining energy homeostasis and emotion during extreme malnourishment.

Restricting-type of anorexia nervosa (AN-R) is a serious disorder affecting adolescents and young adults, and decreases quality of life over long period. Successful weight restoration is an important prognostic factor for disease outcome; however, the underlying mechanism of refeeding-resistance, a core psychopathology relevant to 'ambivalent' eating behaviors, remains unclear in this disorder. Obestatin plays an important role in the regulation of growth hormone release, appetite, and energy metabolism. However, the progress of these patients and changes in the levels of obestatin during treatment were not reported. The purpose of this study was to determine the changes in obestatin levels when energy intake increases in AN-R patients. As a result, obestatin was higher in AN-R patients than in control subjects as well as acyl ghrelin and des-acyl ghrelin. An increase in the intake calorie has decreased obestatin as well as des-acyl ghrelin. These findings indicate that the obestatin is an important factor in the diagnosis and treatment of AN-R, similarly to acyl ghrelin and des-acyl ghrelin. In the future, the research on the clinical application of the ghrelin peptide family and the receptor will be expected to progress.

Lifestyle-related diseases are associated with overeating and lack of exercise. The purpose of this study was to investigate the effect of exercise and high-fat diet on plasma adiponectin and nesfatin levels. Mice were housed for 4 weeks in 4 groups, which included the non-exercise and normal diet (SN), exercise and normal diet (EN), non-exercise and high-fat diet (SF) and the exercise and high-fat diet (EF) group. The mice in the exercise groups were housed in cages with a running wheel and were subjected to voluntary exercise. The food intake (Kcal) of the mice in the exercise groups increased compared to that of the mice in the non-exercise groups (P

Human and mouse urocortin 3 (Ucn3) were first identified in 2001. Ucn3 binds selectively to corticotropin-releasing factor receptor type 2 (CRF-R2). Previous studies have shown that centrally administered Ucn3 decreases food intake in rats. However, the role of Ucn3 in the regulation of gut motility remains to be determined. In the present study, we investigated the effects of peripherally administered Ucn3 on food intake and gastric emptying in mice. After intraperitoneal (i.p.) administration of Ucn3, food intake was measured in the light and dark phases, and the rate of gastric emptying was determined. We found that i.p. administration of Ucn3 significantly inhibited feeding behavior in mice, and significantly delayed gastric emptying 1-2 h after administration in a dose-dependent manner. These results suggest that Ucn3 contributes to the modulation of feeding behavior and gut motility. Thus, Ucn3 and CRF-R2 may be involved in the pathogenesis of functional gastrointestinal and eating disorders.

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, P0.05). 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, P0.01 and des-acyl ghrelin: 300.17 ± 55.95 fmol/ml vs. 107.34 ± 40.63 fmol/ml, P0.05). 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.

Neuromedin S (NMS) was recently identified as an endogenous ligand for the FM-4/TGR-1 receptor in the rat hypothalamus. No previous studies have examined the effect of NMS on gut motility. We examined the effects of intracerebroventricular administration of NMS on food intake in food-deprived and free-feeding mice, and on gastroduodenal motility by using a manometric method, and gastric emptying in mice. We found that NMS decreased food intake and the gastric emptying rate. It also disrupted the motor activity in the antrum and duodenum of conscious food-deprived mice. These results suggest that NMS influences gut motility as well as feeding behavior.

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.

To explore the major etiological features of cholestatic liver disease (CLD) in children, and to investigate the molecular epidemiological distribution of SLC25A13 mutations in CLD.A clinical cross-sectional investigation was performed on 63 CLD cases diagnosed from Oct. 2003 to Mar. 2009 in our department, including 36 males and 27 females. Their clinical data were collected, and etiology and prognosis were analyzed and summarized. Thirteen to 17 mutations in SLC25A13 gene were screened by means of procedures established previously by our group. Several SLC25AJ3 mutations were detected by direct sequencing of DNA fragments amplified by genomic DNA-PCR.No specific etiologies were identified in 24 of the 63 cases. Among the 39 cases with identified etiologies, inherited metabolic diseases were on top of the list, including 6 kinds and 27 cases in total, i.e., neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD, 21 cases), transient galactosemia, tyrosinemia type I, galactose kinase deficiency, ornithine carbamoyl transferase deficiency and glycogen storage disease type I, followed by acquired causes (7 cases in total), such as total parenteral nutrition associated cholestasis (TPNAC), congenital syphilis and CMV hepatitis; and then biliary tract malformation (5 cases in total), including biliary atresia, Caroli's disease and gallbladder polyp, were the third. Ten of the 55 patients on follow-up have passed away, while the remaining 45 cases were improved or recovered clinically. SLC25A13 gene analysis were performed in 44 CLD subjects and 21 of them from 20 families (with 40 SLC25A13 alleles in total) were found to have mutations, and the seven mutations detected were 851-854del (23/40), IVS6 + 5GA (6/40), IVS16ins3kb (3/40), 1638-1660dup (2/30), A541D (1/30), R319X (1/30) and G333D (1/30), respectively, and there were other 3 mutations (3/40) still needing identification in the remaining 3 alleles.The etiologies for CLD in some cases can not be identified. However, inherited metabolic diseases, including NICCD in particular, constitute common causative factors for CLD. Most of the CLD conditions can be improved, even recovered clinically, although some cases presented with poor prognosis. Seven mutations in SLC25A13 gene were detected, among which, 851-854del, IVS6 + 5GA, IVS16ins3kb and 1638-1660dup were the leading four mutations, respectively.

Background Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is a novel inborn error of metabolism due to dysfunction of citrin protein, and much more information about this new disease is still needed for its clinical management. Aims To investigate in detail the clinical and laboratory features of NICCD. Patients 13 NICCD subjects in mainland of China diagnosed in our department since 2006. Methods The anthropometric parameters of the patients at birth were compared with controls, representative biochemical changes and metabolome findings were investigated cross-sectionally, and mutations in the causative gene SLC25A13 were analyzed by protocols established previously. Results The patients showed reduced birth weight, length and ponderal index. Main clinical manifestations consisted of jaundice, hepato/hepatosplenomegaly and steatohepatosis on ultrasonography. Biochemical analysis revealed intrahepatic cholestasis, delayed switch of AFP to albumin, and elevated triglyceride, total cholesterol and LDL-cholesterol together with reduced HDL-cholesterol. Metabolome findings included co-existence of markers for galactosemia and tyrosinemia in urine, and elevated Cit, Met, Thr, Tyr, Lys, Arg and Orn in blood. Mutations of 851-854del, IVS6+5G>A, 1638-1660dup, A541D, IVS16ins3kb, R319X and G333D were detected in the gene SLC25A13 . Conclusions The diagnosis of NICCD cannot be established based just on the numerous but non-specific clinical manifestations and biochemical changes. The relatively specific metabolome features provide valuable tools for its screening and diagnosis, while SLC25A13 mutation analysis should be taken as one of the reliable tools for the definitive diagnosis. The body proportionality at birth, steatohepatosis on ultrasonography, delayed switch of AFP to albumin, dyslipidemia pattern, urinary metabolome features and the novel mutation G333D expanded the clinical spectrum of NICCD.

Citrin deficiency resulting from mutations of the SLC25A13 gene is associated with two major clinical phenotypes; neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and adult-onset type 2 citrullinemia (CTLN2). In Korea, 6 cases of citrin deficiency were diagnosed based on biochemical and molecular findings. Four NICCD patients (2 boys and 2 girls) presented high citrulline levels on a newborn screening test or neonatal cholestasis. They were associated with conjugated hyperbilirubinemia, elevated liver enzymes, hypoalbuminemia, mild hyperammonemia, elevated citrulline, methionine and threonine. All of the hepatic manifestations were resolved spontaneously at the age of 5-9 months. Mutation analysis identified them as compound heterozygotes carrying each of the c.851del4, IVS11+1G>A, IVS13+1G>A, G393S, and IVS16ins3kb mutant alleles. Two adult male CTLN2 patients were identified. They were aged 24 and 37 years, and presented sudden loss of consciousness, hyperammonemia and citrullinemia. They were compound heterozygotes with IVS13+1G>A and IVS16ins3kb, and with c.851del4 and IVS11+1G>A mutant alleles. This report describes the clinical characteristics, biochemical findings and molecular analysis of the SLC25A13 gene of patients with citrin deficiency in Korea.

Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD, MIM#605814) is an inherited metabolic disease resulting from mutations of the gene SLC25A13, which encodes citrin, a liver-type mitochondrial aspartate-glutamate carrier. Mutation analysis is necessary for definitive diagnosis of NICCD patients. So far (March, 2007), 36 kinds of mutation, including 7 nonsense, 10 missense, 11 abnormal splicing, 4 insertion and 4 deletion, have been identified by Kobayashi's group, who cloned the gene in Kagoshima, Japan. To date, most of the NICCD patients reported in the world are Japanese. This study aimed to explore the gene diagnosis procedure of two known SLC25A13 mutations in a pedigree with an NICCD patient from China.DNA was extracted from dried blood spots collected with filter papers from the proband and other 9 members in a NICCD pedigree from China, and then PCR amplification and agarose gel electrophoresis were performed, revealing two mutations preliminarily, which were further proved by Genescan, a procedure established in our laboratory already. Furthermore, the positions and characteristics of the mutations were finally confirmed by DNA sequencing.The proband is a compound heterozygote of two mutations, 851-854del in exon 9 and 1638-1660dup in exon 16 of SLC25A13 gene. His mother and brother carry the former mutation, which predicts a frameshift and introduction of a stop codon at position 286, while his father, one aunt and her son carry the latter, resulting in a frameshift at codon 554, and introducing a stop codon at position 570.A deletion mutation 851-854del in exon 9 and an insertion mutation 1638-1660dup in exon 16 of SLC25A13 gene were identified in the pedigree, providing reliable evidences for both diagnostic confirmation of the patient and the genetic counseling from other members in the pedigree.