Katsuro Iwase

Precise distribution of neuronal nitric oxide synthase mRNA in the rat brain revealed by non-radioisotopic in situ hybridization.

Regional distribution of neurons expressing neuronal nitric oxide synthase mRNA in the rat brain was examined by non-radioisotopic in situ hybridization, using digoxigenin-labeled complementary RNA probes. Clustering of intensely positive neurons was observed in discrete areas including the main and accessory olfactory bulbs, the islands of Calleja, the amygdala, the paraventricular nucleus of the thalamus, several hypothalamic nuclei, the lateral geniculate nucleus, the magnocellular nucleus of the posterior commissure, the superior and inferior colliculi, the laterodorsal and pedunculopontine tegmental nuclei, the nucleus of the trapezoid body, the nucleus of the solitary tract and the cerebellum. Strongly-stained isolated neurons were scattered mainly in the cerebral cortex, the basal ganglia and the brain stem, especially the medulla reticular formation. In the hippocampus, an almost uniform distribution of moderately stained neurons was observed in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the Ammons horn, while more intensely stained isolated neurons were scattered over the entire hippocampal region. These observations can serve as a good basis for studies on function and gene regulation of neuronal nitric oxide synthase.

Hypoglycemia-associated hyperammonemia caused by impaired expression of ornithine cycle enzyme genes in C/EBPalpha knockout mice

Ammonia produced by amino acid metabolism is detoxified through conversion into urea by the ornithine cycle in the liver, whereas carbon skeletons of amino acids are converted to glucose by gluconeogenic enzymes. Promoter and enhancer sequences of several genes for ornithine cycle enzymes interact with members of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family. Disruption of the C/EBPα gene in mice causes hypoglycemia associated with the impaired expression of gluconeogenic enzymes. Here we examined the expression of ornithine cycle enzyme genes in the livers of C/EBPα-deficient mice. mRNA levels for the first, third, fourth, and fifth enzymes of five enzymes in the cycle were decreased in C/EBPα-deficient mice. Protein levels for the first, second, fourth, and fifth enzymes were also decreased. In situhybridization analysis revealed that the enzyme mRNAs were distributed normally in the periportal region but were disordered in C/EBPα-deficient mice with relatively higher mRNA levels in the midlobular region. Blood ammonia concentrations in the mutant mice were severalfold higher than in wild-type mice. Thus, C/EBPα is crucial for ammonia detoxification by ornithine cycle enzymes and for coordination of gluconeogenesis and urea synthesis.

Immunohistochemical localization of arginase II and other enzymes of arginine metabolism in rat kidney and liver.

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were collocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.

Hyperammonemia: regulation of argininosuccinate synthetase and argininosuccinate lyase genes in aggregating cell cultures of fetal rat brain

Hyperammonemia in the brain leads to poorly understood alterations of nitric oxide (NO) synthesis. Arginine, the substrate of nitric oxide synthases, might be recycled from the citrulline produced with NO by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). The regulation of AS and AL genes during hyperammonemia is unknown in the brain. We used brain cell aggregates cultured from dissociated telencephalic cortex of rat embryos to analyze the regulation of AS and AL genes in hyperammonemia. Using RNase protection assay and non-radioactive in situ hybridization on aggregate cryosections, we show that both AS and AL genes are induced in astrocytes but not in neurons of aggregates exposed to 5 mM NH4Cl. Our work suggests that the hyperammonemic brain might increase its recycling of citrulline to arginine.

Time of day and nutrients in feeding govern daily expression rhythms of the gene for sterol regulatory element-binding protein (SREBP)-1 in the mouse liver.

Sterol regulatory element-binding protein-1 (SREBP-1) plays a central role in transcriptional regulation of genes for hepatic lipid synthesis that utilizes diet-derived nutrients such as carbohydrates and amino acids, and expression of SREBP-1 exhibits daily rhythms with a peak in the nocturnal feeding period under standard housing conditions of mice. Here, we report that the Srebp-1 expression rhythm shows time cue-independent and Clock mutation-sensitive circadian nature, and is synchronized with varied photoperiods apparently through entrainment of locomotor activity and food intake. Fasting caused diminution of Srebp-1 expression, while diabetic db/db and ob/ob mice showed constantly high expression with loss of rhythmicity. Time-restricted feedings during mid-light and mid-dark periods exhibited differential effects, the latter causing more severe damping of the oscillation. Therefore, “when to eat in a day (the light/dark cycle),” rather than “whenever to eat in a day,” is a critical determinant to shape the daily rhythm of Srebp-1 expression. We further found that a high-carbohydrate diet and a high-protein diet, as well as a high-fat diet, cause phase shifts of the oscillation peak into the light period, underlining the importance of “what to eat.” Daily rhythms of SREBP-1 protein levels and Akt phosphorylation levels also exhibited nutrient-responsive changes. Taken together, these findings provide a model for mechanisms by which time of day and nutrients in feeding shape daily rhythms of the Srebp-1 expression and possibly a number of other physiological functions with interindividual and interdaily differences in human beings and wild animals subjected to day-by-day changes in dietary timing and nutrients.

The gene for hepatocyte nuclear factor (HNF)‐4α is activated by glucocorticoids and glucagon, and repressed by insulin in rat liver

The gene for a transcription factor hepatocyte nuclear factor‐4α (HNF‐4α) is responsible for maturity‐onset diabetes of the young, type 1. We examined hormonal regulation of the HNF‐4α gene in the liver. Stimulation of primary‐cultured rat hepatocytes with dexamethasone or glucagon led to induction of HNF‐4α mRNA, being antagonized by insulin. In the liver of streptozotocin‐induced diabetic rat, mRNA and protein levels for HNF‐4α were elevated, and were normalized by insulin treatment. Therefore, HNF‐4α in the liver is likely to be involved in the regulation of glucose metabolism in response to these hormones.

Relationship Between Pancreatic Secretory Trypsin Inhibitor and Early Recurrence of Intrahepatic Cholangiocarcinoma Following Surgical Resection

OBJECTIVES:The extremely unfavorable prognosis of intrahepatic cholangiocarcinoma (ICC), even after surgical resection, is mainly attributed to a high rate of recurrence. The aim of this study was to identify the molecules associated with early recurrence of ICC following resection.METHODS:Between December 1984 and July 2003, 46 patients with ICC underwent surgical resection. The clinical outcome of these patients was evaluated in view of the time of recurrence. Consequently, we categorized ICC patients into subgroups, based on the clinical results, and screened differentially expressed genes by DNA microarray analysis. Furthermore, the obtained results were validated in an independent sample set by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemistry was performed to assess the expressed genes at the protein level.RESULTS:The survival of patients with early recurrence, occurring within a year after surgical resection, was significantly poor after surgery and even after recurrence, as compared to that of patients whose recurrence occurred beyond a year after surgery.By the DNA microarray analysis, 13 differentially expressed genes were picked up, and quantitative RT-PCR reaction identified the pancreatic secretory trypsin inhibitor (PSTI) as a candidate gene associated with early recurrence of ICC after resection. This observation was confirmed through examination of an independent set of samples, in which the patients with higher levels of PSTI mRNA expression had significantly shorter recurrence-free survival. Immunohistochemically, PSTI was expressed in the cytoplasm of cancer cells.CONCLUSIONS:PSTI might be a potential marker for identifying ICC patients with an increased risk of early recurrence after surgical resection.

CCAAT/enhancer-binding protein β is required for activation of genes for ornithine cycle enzymes by glucocorticoids and glucagon in primary-cultured hepatocytes

Transcription of genes for enzymes of the ornithine cycle is activated by hormones such as glucocorticoids and glucagon. Promoters and enhancers of several genes for the enzymes interact with the CCAAT/enhancer‐binding protein (C/EBP) family of transcription factors, and C/EBPβ has been suggested to mediate glucocorticoid response of the gene for arginase, the last enzyme of the cycle. To determine the contribution of C/EBPβ to hormonal regulation of genes for ornithine cycle enzymes, we examined mice with targeted disruption of the C/EBPβ gene. Induction of genes for the enzymes by intraperitoneal injection of dexamethasone and glucagon was almost intact in the liver of C/EBPβ‐deficient mice. On the other hand, in primary‐cultured hepatocytes derived from C/EBPβ‐deficient mice, induction of genes for the first enzyme carbamylphosphate synthetase, as well as for arginase, in response to dexamethasone and/or glucagon was severely impaired. Therefore, C/EBPβ is required for hormonal induction of the genes for ornithine cycle enzymes in primary‐cultured hepatocytes, while the deficiency of C/EBPβ is compensated for in vivo.

Expression of citrulline–nitric oxide cycle in lipopolysaccharide and cytokine-stimulated rat astroglioma C6 cells

Nitric oxide (NO) is involved in many physiological and pathological processes in the brain. NO is synthesized from arginine by nitric oxide synthase (NOS), with citrulline generated as a by-product of the reaction. Thus, citrulline can by recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) via the citrulline-NO cycle. Rat astroglioma C6 cells were treated with bacterial lipopolysaccharide (LPS), interferon-gamma (IFNgamma) and tumor necrosis factor-alpha, and the expression of the enzymes of the citrulline-NO cycle was investigated by RNA blot and immunoblot analyses. NO production from arginine and citrulline was also assessed. iNOS mRNA and protein were induced 6-12 h after stimulation with LPS and cytokines and decreased at 24 h. AS mRNA increased up to 12 h and decreased at 24 h. AS protein increased gradually up to 48 h. On the other hand, AL mRNA remained unchanged by stimulation. NO production from arginine was enhanced by the treatment with LPS and cytokines. NO production was also observed when arginine was replaced by citrulline. These results indicate that NO production is enhanced in LPS- and cytokine-stimulated C6 cells due to induction of iNOS and that the citrulline-arginine recycling is important for NO production.

Multifactorial Regulation of Daily Rhythms in Expression of the Metabolically Responsive Gene Spot14 in the Mouse Liver

Spot14 is a putative transcriptional regulator for genes involved in fatty acid synthesis. The Spot14 gene is activated in response to lipogenic stimuli such as dietary carbohydrate and is also under circadian regulation. The authors investigated factors responsible for daily oscillation of Spot14 expression. If mice were kept under a 12-h light/12-h dark cycle with ad libitum feeding, Spot14 mRNA levels in the liver reached a peak at an early dark period when mice, as nocturnal animals, start feeding. Under fasting, while Spot14 mRNA levels were generally decreased, the rhythmicity was still maintained, suggesting contribution of both nutritional elements and circadian clock factors on robust rhythmicity of Spot14 expression. Effects of circadian clock factors were confirmed by the observations that the circadian rhythm of Spot14 expression was seen also under the constant darkness and that the rhythmicity was lost in Clock mutant mice. When mice were housed in short-photoperiod (6-h light/18-h dark) and long-photoperiod (18-h light/6-h dark) cycles, rhythms of Spot14 mRNA levels were phase advanced and phase delayed, respectively, being concordant with the notion that Spot14 expression is under the control of the light-entrainable oscillator. As for nutritional mediators, in the liver of db/db mice exhibiting hyperinsulinemia-accompanied hyperglycemia, Spot14 mRNA levels were constantly high without apparent rhythmicity, consistent with previous observations for strong activation of the Spot14 gene by glucose and insulin. Restricted feeding during the 4-h mid-light period caused a phase advance of the Spot14 expression rhythm. On the other hand, restricted feeding during the 4-h mid-dark period led to damping of the rhythmicity, apparently resulting from the separation of phases between effects of the light/dark cycle and feeding on Spot14 expression. Thus, the daily rhythm of Spot14 expression in the liver is under the control of the light-entrainable oscillator, food-entrainable oscillator, and food-derived nutrients, in a separate or cooperative manner.