A case of infective endocarditis in an 8-year-old boy 3 months after transcatheter atrial septal defect closure using Figulla Flex II occluder.

Infective endocarditis is a rare complication of atrial septal defect closure using transcatheter procedure. We report about infective endocarditis in an 8-year-old boy 3 months after transcatheter closure using a Figulla Flex II atrial septal defect occluder. Transesophageal echocardiography showed vegetation attached to the left atrium side of the device. Device removal and atrial septal defect closure were performed. The device was less endothelialized on the left than on the right atrium side. Therefore, insufficient endothelialization may cause infective endocarditis.

Analysis of the Stimulative Effect of Tryptophan on Hepatic Protein Synthesis in Rats.

Tryptophan is an essential amino acid important as a protein building block, but it also serves as substrate for the generation of several bioactive compounds with important physiological roles. Furthermore, tryptophan has been reported to have a unique role as a nutritional signaling molecule that regulates protein synthesis in mouse and rat liver. In the present study, the acute effects of tryptophan on protein synthesis were confirmed and compared with those of leucine in rats. Eighteen hours fasted rats were orally administered of tryptophan or leucine at a dose of 135 mg/100 g body weight by gavage and then sacrificed 1 h after administration. The effects of tryptophan and leucine on the rate of protein synthesis were evaluated by the surface sensing of translation (SUnSET) method. We also examined the ability of tryptophan to induce activation of the mTOR pathway by measuring phosphorylation of 4E-BP1 and S6K1. Oral administration of tryptophan led to a stimulation of the rate of protein synthesis concomitant with activation of mTOR pathway in the liver, but not in skeletal muscle. We also investigated the sensitivity of liver protein synthesis to tryptophan administration. The half-maximal effective doses (ED50) of tryptophan in stimulating 4E-BP1 and S6K1 phosphorylation were both about 60% of daily intake. The effect of tryptophan on hepatic protein synthesis was similar to that of leucine on muscle protein synthesis, and the sensitivity of liver protein synthesis to tryptophan administration appeared to be almost the same or slightly lower than that of muscle protein synthesis to leucine administration.

Essential Amino Acid Intake Is Required for Sustaining Serum Insulin-like Growth Factor-I Levels but Is Not Necessarily Needed for Body Growth.

Essential amino acids (EAAs) are those that cannot be synthesized enough to meet organismal demand; therefore, it is believed that they must be taken from the diet for optimal growth. The growth hormone (GH)/insulin-like growth factor-I (IGF-I) system is also considered significant for growth regulation in mammals. This study aimed to evaluate the relative contributions of protein nutrition and the GH/IGF-I system to body growth regulation. Experiments using rodents and hepatocyte-derived cell lines subjected to EAA deficiency showed that a reduction in the serum EAA concentration hinders Igf1 transcription in the liver in a cell-autonomous manner, thereby decreasing serum IGF-I levels. Remarkably, when the serum IGF-I level of mice on a low-protein diet was restored by the recombinant IGF-I infusion, the body growth was mostly rescued, although the mice were still deficient in EAA intake. Meanwhile, the GH signal activation and subsequent Igf1 transcription were also dramatically diminished by EAA deprivation in the cell culture model. Altogether, we demonstrate that EAAs are not strictly necessary for animal growth as building blocks but are required as IGF-I-tropic cues. The results will bring a paradigm shift regarding the definition of "essential" amino acids.

Successful treatment of symptomatic first-degree atrioventricular block after Occlutech Figulla Flex II atrial septal defect occluder placement.

Given the rarity of significant first-degree atrioventricular block (AVB) after Occlutech Figulla Flex II atrial septal defect occluder (OFF II, Occlutech International AB, Helsingborg, Sweden) placement, its management and predictors of recovery have not been established. We report a 9-year-old boy who developed significant first-degree AVB a day after OFF II deployment. Thereafter, oral prednisolone was initiated at 1.5 mg/kg/day. On day 2 of steroid treatment, normal sinus rhythm without conduction delays was occasionally observed, while the prolonged PR interval on the exercise stress test was ameliorated. After 5 days of steroid administration, normal sinus rhythm without conduction delays was completely achieved. An increase in the frequency of normal sinus rhythm and the absence of conduction delays on electrocardiogram monitoring and exercise stress test play an important role in predicting recovery from prolonged PR interval. Thus, steroid treatment can be effective and should be considered for patients who develop first-degree AVB after undergoing OFF II placement for transcatheter device closure of atrial septal defect. .

Coronary artery aneurysms of unknown origin in a 14-year-old girl.

The case of a 14-year-old girl with acute myocardial infarction due to coronary artery aneurysms (CAAs) of unknown origin, which resembled coronary artery lesions caused by Kawasaki disease, is reported. She was transferred to our hospital due to chest pain with ST-T elevation. She had no history of Kawasaki disease. On the first admission, she was misdiagnosed with acute myocarditis. Then, 54 days later, she experienced chest pain with exertional dyspnea. Her electrocardiogram showed negative T waves in the chest leads. A CAA of the left coronary artery was suspected on two-dimensional echocardiography. Coronary angiograms showed 90% stenosis and multiple CAAs of the left anterior descending artery and the bifurcation of the left coronary artery. Both the right coronary artery and left circumflex artery were occluded. A left ventriculogram showed dyskinesis and an aneurysm at the apex. She underwent triple-vessel coronary artery bypass grafting, and her symptoms improved. In addition, an intracranial aneurysm was also found on cerebral angiography. There were no specific laboratory findings other than SS-A antibodies. It was suspected that the weakness of the vessels was related to the disease. It may have been a different disease that was never previously detected, but her CAAs were Kawasaki-like CAAs.

Low-arginine and low-protein diets induce hepatic lipid accumulation through different mechanisms in growing rats.

BACKGROUND: Dietary protein deficiency and amino acid imbalance cause hepatic fat accumulation. We previously demonstrated that only arginine deficiency or total amino acid deficiency in a diet caused significant hepatic triglyceride (TG) accumulation in young Wistar rats. In this study, we explored the mechanisms of fatty liver formation in these models. METHODS: We fed 6-week-old male Wistar rats a control diet (containing an amino acid mixture equivalent to 15% protein), a low-total-amino acid diet (equivalent to 5% protein; 5PAA), and a low-arginine diet (only the arginine content is as low as that of the 5PAA diet) for 2 weeks. RESULTS: Much greater hepatic TG accumulation was observed in the low-arginine group than in the low-total-amino acid group. The lipid consumption rate and fatty acid uptake in the liver did not significantly differ between the groups. In contrast, the low-total-amino acid diet potentiated insulin sensitivity and related signaling in the liver and enhanced de novo lipogenesis. The low-arginine diet also inhibited hepatic very-low-density lipoprotein secretion without affecting hepatic insulin signaling and lipogenesis. CONCLUSIONS: Although the arginine content of the low-arginine diet was as low as that of the low-total-amino acid diet, the two diets caused fatty liver via completely different mechanisms. Enhanced lipogenesis was the primary cause of a low-protein diet-induced fatty liver, whereas lower very-low-density lipoprotein secretion caused low-arginine diet-induced fatty liver.

Disruption of insulin receptor substrate-2 impairs growth but not insulin function in rats.

Insulin receptor substrate (IRS)-2, along with IRS-1, is a key signaling molecule that mediates the action of insulin and insulin-like growth factor (IGF)-I. The activated insulin and IGF-I receptors phosphorylate IRSs on tyrosine residues, leading to the activation of downstream signaling pathways and the induction of various physiological functions of insulin and IGF-I. Studies using IRS-2 knockout (KO) mice showed that the deletion of IRS-2 causes type 2 diabetes due to peripheral insulin resistance and impaired ß-cell function. However, little is known about the roles of IRS-2 in other animal models. Here, we created IRS-2 KO rats to elucidate the physiological functions of IRS-2 in rats. The body weights of IRS-2 KO rats at birth were lower compared with those of their WT littermates. The postnatal growth of both male and female IRS-2 KO rats was also suppressed. Compared with male WT rats, the glucose and insulin tolerance of male IRS-2 KO rats were slightly enhanced, whereas a similar difference was not observed between female WT and IRS-2 KO rats. Besides the modestly increased insulin sensitivity, male IRS-2 KO rats displayed the enhanced insulin-induced activation of the mTOR complex 1 pathway in the liver compared with WT rats. Taken together, these results indicate that in rats, IRS-2 plays important roles in the regulation of growth but is not essential for the glucose-lowering effects of insulin.

Growth hormone increases regulator of calcineurin 1-4 (Rcan1-4) mRNA through c-JUN in rat liver.

Growth hormone (GH) activates multiple signal transduction pathways. To investigate these pathways, we identified novel genes whose transcription was induced by GH in the liver of hypophysectomized (HPX) rats using the suppression subtractive hybridization technique. We found that regulator of calcineurin 1 (Rcan1) mRNA was upregulated by GH administration. RCAN1 regulates the activity of calcineurin, a Ca/calmodulin-dependent phosphatase. Rcan1 encodes two major transcripts, Rcan1-1 and Rcan1-4, resulting from differential promoter use and first exon choice. We found that a single injection of GH increased the levels of Rcan1-4 mRNA and RCAN1-4 protein transiently, but did not increase Rcan1-1 mRNA in HPX rat liver. Then the molecular mechanism of GH to induce Rcan1-4 transcription was examined in rat hepatoma H4IIE cells. Experiments using inhibitors suggested that c-JUN N-terminal kinase was required for the induction of Rcan1-4 mRNA by GH. GH increased the levels of phosphorylated c-JUN protein and c-Jun mRNA in HPX rat liver. The luciferase and electrophoretic mobility shift assays showed that c-JUN upregulated Rcan1-4 mRNA by binding to the cAMP-responsive element in the upstream of Rcan1 exon 4. These results indicate that GH activates c-JUN to affect the activity of calcineurin by the induction of Rcan1-4 in rat liver.

A translation repressor, 4E-BP1, regulates the triglyceride level in rat liver during protein deprivation.

Protein deprivation has been shown to induce fatty liver in humans and animals, but the molecular mechanisms underlying such induction are largely unknown. Our previous studies have shown that a low-protein diet increases eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) protein and triglyceride (TG) levels in rat liver. 4E-BP1 is known to repress translation by binding to eIF4E. There is also evidence indicating that 4E-BP1 regulates lipid metabolism. Here, we examined the role of 4E-BP1 on TG accumulation in the livers of rats under protein deprivation. The low-protein diet rapidly increased the hepatic 4E-BP1 mRNA level within 1 day, followed by the induction of hepatic TG accumulation. The knockdown of hepatic 4E-BP1 attenuated the TG accumulation in rat liver induced by the low-protein diet. 4E-BP1 knockdown also increased the protein level of carnitine palmitoyltransferase 1A (CPT1A), a regulator of fatty acid oxidation, in the liver of rats fed a low-protein diet. These results indicate that a low-protein diet increases the amount of 4E-BP1, leading to TG accumulation in rat liver. We thus conclude that 4E-BP1 plays an important role in inducing hepatic steatosis under protein deprivation.

Growth hormone activates X-box binding protein 1 in a sexually dimorphic manner through the extracellular signal-regulated protein kinase and CCAAT/enhancer-binding protein ß pathway in rat liver.

Growth hormone (GH) has multiple physiological roles, acting on many organs. In order to investigate its roles in rat liver, we tried to identify novel genes whose transcription was regulated by GH. We identified X-box binding protein 1 (Xbp1) as a candidate gene. XBP1 is a key transcription factor activated in response to endoplasmic reticulum (ER) stress. The purpose of this study was to investigate the mode of action of GH on XBP1, including the relation with ER stress, sex-dependent expression of the mRNA, and the signaling pathway. Intravenous administration of GH rapidly and transiently increased Xbp1 mRNA in hypophysectomized rat livers. Neither phosphorylated inositol-requiring-1α (IRE1α) nor phosphorylated PKR-like ER kinase (PERK) increased, suggesting that Xbp1 expression is induced by an ER stress-independent mechanism. The active form of XBP1(S) protein was increased by GH administration and was followed by an increased ER-associated dnaJ protein 4 (ERdj4) mRNA level. XBP1(S) protein levels were predominantly identified in male rat livers with variations among individuals similar to those of phosphorylated signal transducer and activator of transcription 5B (STAT5B), suggesting that XBP1(S) protein levels are regulated by the sex-dependent secretary pattern of GH. The GH signaling pathway to induce Xbp1 mRNA was examined in rat hepatoma H4IIE cells. GH induced the phosphorylation of CCAAT/enhancer-binding protein ß (C/EBPß) following extracellular signal-regulated protein kinase (ERK) phosphorylation. Taken together, the results indicated that XBP1 is activated by GH in rat liver in a sexually dimorphic manner via ERK and C/EBPß pathway.

Low-protein diet enhances adiponectin secretion in rats.

Previous studies including ours have shown that a low-protein diet up-regulates insulin signaling in the liver and muscle and induces fatty liver in rats. Adiponectin is known as an insulin-sensitizing adipocytokine. We, therefore, examined the effect of a low-protein diet on the adiponectin levels in rats. The low-protein diet significantly increased serum adiponectin level. However, mRNA and protein levels of adiponectin in white adipose tissue (WAT) were not changed by the low-protein diet. Since it is known that oligomerization is important to control serum adiponectin level, we examined the population of adiponectin oligomeric forms in WAT and found that low-protein diet did not change it. Despite these events, the amount of its secretion was significantly increased in the adipocytes isolated from WAT of low-protein diet-fed rats. These results indicate that a low-protein diet enhances adiponectin secretion, which is not due to the increased intracellular amount and oligomerization of adiponectin.

Positive pediatric exercise capacity trajectory predicts better adult Fontan physiology rationale for early establishment of exercise habits.

OBJECTIVE: Exercise training is recommended for its possible favorable effects on Fontan pathophysiology. This study aimed to elucidate the impact of pediatric exercise capacity trajectory, which may mimic the effect of exercise training, on late adult Fontan pathophysiology. METHODS: Since 1990, 97 Fontan patients had consecutively undergone two serial cardiopulmonary exercise tests (CPX1 and CPX2) during childhood (ages 8 ±â€¯2 and 14 ±â€¯2 years) and one during adulthood (CPX3; age 23 ±â€¯5 years). The changes in peak oxygen uptake (PVO2: % of normal value) from CPX1 to CPX2 (1-dPVO2) and from CPX2 to CPX3 (2-dPVO2) were calculated, and then the patients were divided into four subgroups according the 1-dPVO2 and 2-dPVO2. RESULTS: In their adulthood, when compared with groups with negative 1-dPVO2, the central venous pressure, plasma brain natriuretic peptide level, and renal resistive index were lower, whereas liver synthetic function, body fat-free percentage, and PVO2 were higher in those with positive 1-dPVO2 (p < 0.05-0.0001). However, these favorable associations of 2-d-PVO2 with adult Fontan pathophysiology were not observed, except for the PVO2. After CPX3, 13 unexpected events occurred, and the risk was 76% lower in the groups having positive 1-dPVO2 than in those with negative 1-dPVO2 (hazard ratio, 0.24; 95% confidence interval, 0.09-0.62; p = 0.0035). CONCLUSIONS: A positive exercise capacity trajectory during childhood predicts better adult Fontan pathophysiology, including better prognosis. Thus, prescription of exercise could be a promising strategy in the management of pediatric Fontan patients.

Cellular fraction analysis of pericardial effusion helps the diagnosis of eosinophilic myocarditis.

Eosinophilic myocarditis is rare in children, and consequently, it is difficult to diagnose eosinophilic myocarditis rapidly. We report the clinical course of acute eosinophilic myocarditis with pericarditis in two adolescent boys and their associated electrocardiograms. The two patients, 13- and 14-year-old boys, developed cardiomegaly and chest pain with vomiting. On examination by two-dimensional echocardiography, thickening of the ventricular septum and a pericardial effusion were detected. The eosinophil count had increased by the pericardial effusion. Acute eosinophilic myocarditis often complicates a moderate to severe pericardial effusion owing to acute pericarditis. A cellular fraction analysis of the pericardial effusion is easy and useful for the diagnosis of eosinophilic myocarditis. Some serial changes in the electrocardiogram occur during each stage of acute eosinophilic myocarditis. They are induced by eosinophilic granules, which are capable of inducing tissue damage and dysfunction, and those changes in the electrocardiogram resemble the changes after an acute myocardial infarction. It is important to know the characteristics of eosinophilic myocarditis in order to prevent lethal complications.

Importance of Serum Amino Acid Profile for Induction of Hepatic Steatosis under Protein Malnutrition.

We previously reported that a low-protein diet caused animals to develop fatty liver containing a high level of triglycerides (TG), similar to the human nutritional disorder "kwashiorkor". To investigate the underlying mechanisms, we cultured hepatocytes in amino acid-sufficient or deficient medium. Surprisingly, the intracellular TG level was increased by amino acid deficiency without addition of any lipids or hormones, accompanied by enhanced lipid synthesis, indicating that hepatocytes themselves monitored the extracellular amino acid concentrations to induce lipid accumulation in a cell-autonomous manner. We then confirmed that a low-amino acid diet also resulted in the development of fatty liver, and supplementation of the low-amino acid diet with glutamic acid to compensate the loss of nitrogen source did not completely suppress the hepatic TG accumulation. Only a dietary arginine or threonine deficiency was sufficient to induce hepatic TG accumulation. However, supplementation of a low-amino acid diet with arginine or threonine failed to reverse it. In silico analysis succeeded in predicting liver TG level from the serum amino acid profile. Based on these results, we conclude that dietary amino acid composition dynamically affects the serum amino acid profile, which is sensed by hepatocytes and lipid synthesis was activated cell-autonomously, leading to hepatic steatosis.

Branched-chain amino acid supplementation restores reduced insulinotropic activity of a low-protein diet through the vagus nerve in rats.

BACKGROUND: Previously, we reported that a low-protein diet significantly reduced insulin secretion in response to feeding within 1 h in rats, suggesting that the insulinotropic effect of dietary protein plays an important role in maintaining normal insulin release. The current study aimed to elucidate whether deficiency of certain amino acids could diminish the insulinotropic activity and to investigate whether reduced insulin secretion in response to a low-protein diet is restored by supplementation with certain amino acids. METHODS: First, we fed male Wistar rats (5-6 rats per group) with diets deficient in every single amino acid or three branched-chain amino acids (BCAAs); within 1-2 h after the onset of feeding, we measured the plasma insulin levels by using an enzyme-linked immunosorbent assay (ELISA). As insulin secretion was reduced in BCAA-deficient groups, we fed low-protein diets supplemented with BCAAs to assess whether the reduced insulin secretion was restored. In addition, we treated the pancreatic beta cell line MIN6 with BCAAs to investigate the direct insulinotropic activity on beta cells. Lastly, we investigated the effect of the three BCAAs on sham-operated or vagotomized rats to assess involvement of the vagus nerve in restoration of the insulinotropic activity. RESULTS: Feeding a low-protein diet reduced essential amino acid concentrations in the plasma during an absorptive state, suggesting that reduced plasma amino acid levels can be an initial signal of protein deficiency. In normal rats, insulin secretion was reduced when leucine, valine, or three BCAAs were deficient. Insulin secretion was restored to normal levels by supplementation of the low-protein diet with three BCAAs, but not by supplementation with any single BCAA. In MIN6 cells, each BCAA alone stimulated insulin secretion but the three BCAAs did not show a synergistic stimulatory effect. The three BCAAs showed a synergistic stimulatory effect in sham-operated rats but failed to stimulate insulin secretion in vagotomized rats. CONCLUSIONS: Leucine and valine play a role in maintaining normal insulin release by directly stimulating beta cells, and supplementation with the three BCAAs is sufficient to compensate for the reduced insulinotropic activity of the low-protein diet, through the vagus nerve.

MELAS syndrome associated with a new mitochondrial tRNA-Val gene mutation (m.1616A>G).

We describe the case of a 40-year-old-man with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, with cardiomyopathy and severe heart failure. He had a mitochondrial transfer RNA (tRNA) mutation (m.1616A>G) of the (tRNA-Val) gene, and it was not found in MELAS syndrome ever before. The presence of this newly observed tRNA-Val mutation (m.1616A>G) may induce multiple respiratory chain enzyme deficiencies and contribute to MELAS syndrome symptoms that are associated with mitochondrial DNA (mtDNA) mutations. We report that the pathognomonic symptom in MELAS syndrome caused by this newly observed mtDNA mutation may be rapid progression of cardiomyopathy and severe heart failure.

Triglyceride synthesis in hepatocytes isolated from rats fed a low-protein diet is enhanced independently of upregulation of insulin signaling.

It is known that protein malnutrition develops fatty liver in rats. However, the mechanisms by which protein malnutrition enhances lipid accumulation in the liver are not fully understood. Our previous studies have demonstrated that protein malnutrition upregulates insulin signaling with an increase in TG levels in rat livers. Here, we examined whether the upregulated insulin signaling contributes to an enhancement of TG accumulation under protein malnutrition. As it is difficult to analyze insulin-induced hepatic TG synthesis in vivo, the isolated hepatocytes derived from rats fed a low-protein diet were used. The hepatocytes were isolated from rats fed a 15% casein diet (15C) as a control diet or a 5% casein diet (5C) as a low-protein diet and then treated with insulin. As shown in vivo, insulin signaling was upregulated in isolated hepatocytes from 5C-fed rats (5C hepatocytes). However, the insulin-induced increase in the mRNA levels of lipogenic enzymes, including acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS), was similar in both groups. The amounts of TG synthesized from both glucose and palmitate, as well as ACC1 and FAS protein levels, were increased at the basal state in 5C hepatocytes, but were not further increased by insulin. These results indicate that TG synthesis via both de novo fatty acid synthesis and esterification is enhanced in 5C hepatocytes, which is independent of the upregulation of insulin signaling.

A Rare Coincidence of Sitosterolemia and Familial Mediterranean Fever Identified by Whole Exome Sequencing.

Whole exome sequencing (WES) technologies have accelerated genetic studies of Mendelian disorders, yielding approximately 30% diagnostic success. We encountered a 13-year-old Japanese female initially diagnosed with familial hypercholesterolemia on the basis of clinical manifestations of severe hypercholesterolemia (initial LDL cholesterol=609 mg/dl at the age of one) and systemic intertriginous xanthomas with histories of recurrent self-limiting episodes of fever and arthritis. Both her phenotypes seemed to co-segregate in a recessive manner. We performed WES on this patient, who was considered a proband. Among 206,430 variants found in this individual, we found 18,220 nonsense, missense, or splice site variants, of which 3,087 were rare (minor allele frequency ≤ 0.01 or not reported) in 1000 Genome (Asian population). Filtering by assuming a recessive pattern of inheritance with the use of an in silico annotation prediction tool, we successfully narrowed down the candidates to the compound heterozygous mutations in the ABCG5 gene (c.1256G＞A or p.Arg419His/c.1763-1G＞A [splice acceptor site]) and to the double-compound heterozygous mutations in the MEFV gene (c.329T＞C/C or p.Leu110Pro/c.442G＞C/C or p.Glu148Val). The patient was genetically diagnosed with sitosterolemia and familial Mediterranean fever using WES for the first time. Such a comprehensive approach is useful for identifying causative mutations for multiple unrelated inheritable diseases.