Naoto Omata

Characterization of acetate metabolism in tumor cells in relation to cell proliferation: Acetate metabolism in tumor cells

To reveal the metabolic fate of acetate in neoplasms that may characterize the accumulation patterns of [1-(11)C]acetate in tumors depicted by positron emission tomography. Four tumor cell lines (LS174T, RPMI2650, A2780, and A375) and fibroblasts in growing and resting states were used. In uptake experiments, cells were incubated with[1-(14)C]acetate for 40 min. [(14)C]CO(2) was measured in the tight-air chamber, and the metabolites in cells were identified by thin layer chromatography and paper chromatography. The glucose metabolic rate of each cell line was measured with [2,6-(3)H]2-deoxy-glucose (DG), and the growth activity of each cell line was estimated by measuring the incorporation of [(3)H]methyl thymidine into DNA. Compared with resting fibroblasts, all four tumor cell lines showed higher accumulation of (14)C activity from [1-(14)C]acetate. These tumor-to-normal ratios of [1-(14)C]acetate were larger than those of DG. Tumor cells incorporated (14)C activity into the lipid-soluble fraction, mostly of phosphatidylcholine and neutral lipids, more prominently than did fibroblasts. The lipid-soluble fraction of (14)C accumulation in cells showed a positive correlation with growth activity, whereas the water-soluble and CO(2) fractions did not. These findings suggest that the high tumor-to-normal ratio of [1-(14)C]acetate is mainly due to the enhanced lipid synthesis, which reflects the high growth activity of neoplasms. This in vitro study suggests that [1-(11)C]acetate is appropriate for estimating the growth activity of tumor cells.

Plasma levels of adiponectin and tumor necrosis factor-alpha in patients with remitted major depression receiving long-term maintenance antidepressant therapy

Adiponectin, an adipose tissue-specific plasma protein, is involved in insulin sensitization and has anti-atherosclerotic properties, whereas tumor necrosis factor-alpha (TNF-alpha), a pro-inflammatory protein, plays important roles in inflammatory endothelial injury and atherosclerotic changes. It has been reported that adiponectin and TNF-alpha inhibit each others expression and production in adipocytes. Several in vitro studies indicated that antidepressant medications decreased the production of pro-inflammatory cytokines including TNF-alpha, but the effect of antidepressants on the expression of adiponectin is still unknown. We examined the plasma levels of TNF-alpha and adiponectin in patients with remitted depression receiving maintenance antidepressant therapy for longer than half a year, and compared the levels with those in healthy controls. The plasma levels of TNF-alpha and adiponectin in the remitted depression group were significantly lower and higher than those in the control group, respectively. This preliminary cross-sectional study suggests the possibility that maintenance antidepressant therapy may have anti-inflammatory effects and prevent the development of atherosclerosis.

Neuroprotective effect of chronic lithium treatment against hypoxia in specific brain regions with upregulation of cAMP response element binding protein and brain-derived neurotrophic factor but not nerve growth factor: comparison with acute lithium treatment.

OBJECTIVES We evaluated the neuroprotective effect of chronically or acutely administered lithium against hypoxia in several brain regions. Furthermore, we investigated the contribution of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and cAMP response element binding protein (CREB) to the neuroprotective effect of lithium. METHODS Brain slices were prepared from rats that had been treated chronically or acutely with lithium. The cerebral glucose metabolic rate (CMRglc) before and after hypoxia loading to brain slices was measured using the dynamic positron autoradiography technique with [(18)F]2-fluoro-2-deoxy-D-glucose. The changes of expression of proteins were investigated using Western blot analysis. RESULTS Before hypoxia loading, the CMRglc did not differ between the lithium-treated and untreated groups. After hypoxia loading, the CMRglc of the untreated group was significantly lower than that before hypoxia loading. However, the CMRglc of the chronic lithium treatment group recovered in the frontal cortex, caudate putamen, hippocampus and cerebellum, but not in the thalamus. In contrast, the CMRglc of the acute lithium treatment group did not recover in any analyzed brain regions. After chronic lithium treatment, the levels of expression of BDNF and phospho-CREB were higher than those of untreated rats in the frontal cortex, but not in the thalamus. However, the expression of NGF did not change in the frontal cortex and thalamus. CONCLUSIONS These results demonstrated that lithium was neuroprotective against hypoxia only after chronic treatment and only in specific brain regions, and that CREB and BDNF might contribute to this effect.

Hypoxic but not ischemic neurotoxicity of free radicals revealed by dynamic changes in glucose metabolism of fresh rat brain slices on positron autoradiography.

Dynamic changes in the regional cerebral glucose metabolic rate induced by hypoxia/reoxygenation or ischemia/reperfusion were investigated with a positron autoradiography technique. Fresh rat brain slices were incubated with [18F]2-fluoro-2-deoxy-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution at 36°C, and serial two-dimensional time-resolved images of [18F]FDG uptake in the slices were obtained. In the case of loading hypoxia (oxygen deprivation)/pseudoischemia (oxygen and glucose deprivation) for various periods of time, the net influx constant (K) of [18F]FDG at preloading and after reoxygenation/pseudoreperfusion (post-loading) was quantitatively evaluated by applying the Patlak graphical method to the image data. Regardless of the brain region, with hypoxia lasting ≥20 minutes, the postloading K value was decreased compared with the unloaded control, whereas with pseudoischemia of ≤40 minutes, approximately the same level as the unloaded control was maintained. Next, the neuroprotective effect against hypoxia/pseudoischemia loading induced by the addition of a free radical scavenger or an N-methyl-D-aspartate (NMDA) antagonist was assessed by determining whether a decrease in the postloading K value was prevented. Whereas with 20-minute hypoxia, both agents exhibited a neuroprotective effect, in the case of 50-minute pseudoischemia, only the NMDA antagonist did so, with the free radical scavenger being ineffective. These results demonstrate that hypoxia causes irreversible neuronal damage within a shorter period than ischemia, with both free radicals and glutamate suggested to be involved in tandem in the neurotoxicity induced by hypoxia, whereas glutamate alone is involved in ischemic neurotoxicity.

Dynamic changes in glucose metabolism of living rat brain slices induced by hypoxia and neurotoxic chemical-loading revealed by positron autoradiography.

Summary. Fresh rat brain slices were incubated with 2-deoxy-2-[18F]-fluoro-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution at 36°C, and serial two-dimensional time-resolved images of [18F]FDG uptake were obtained from these specimens on imaging plates. The fractional rate constant (= k3*) of [18F]FDG proportional to the cerebral glucose metabolic rate (CMRglc) was evaluated by applying the Gjedde-Patlak graphical method to the image data. With hypoxia loading (oxygen deprivation) or glucose metabolism inhibitors acting on oxidative phosphorylation, the k3* value increased dramatically suggesting enhanced glycolysis. After relieving hypoxia ≤10-min, the k3* value returned to the pre-loading level. In contrast, with ≥20-min hypoxia only partial or no recovery was observed, indicating that irreversible neuronal damage had been induced. However, after loading with tetrodotoxin (TTX), the k3* value also decreased but returned to the pre-loading level even after 70-min TTX-loading, reflecting a transient inhibition of neuronal activity. This technique provides a new means of quantifying dynamic changes in the regional CMRglc in living brain slices in response to various interventions such as hypoxia and neurotoxic chemical-loading as well as determining the viability and prognosis of brain tissues.

Dynamic changes in glucose metabolism by lactate loading as revealed by a positron autoradiography technique using rat living brain slices

To demonstrate the preference of lactate over glucose as an energy substrate in normal brain tissue under normoxic condition, the dynamic changes in glucose uptake by lactate loading were investigated in living rat brain slices using a positron autoradiography technique. Fresh rat brain slices were incubated with [18F]2-fluoro-2-deoxy-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution containing 10 mM glucose at 36 degrees C. During incubation, serial two-dimensional imaging of [18F]FDG uptake in the slices was constructed on the imaging plates. Lactate loading (20 mM) reversibly suppressed the [18F]FDG accumulation up to 80 min. Compared with the pre-loading and the unloaded control values, [18F]FDG uptake was suppressed to 25-45% in cerebral regions and 6-7% in cerebellum. The lactate concentration in the surrounding medium decreased after lactate loading. Hence brain tissue preferentially uses lactate over glucose under normoxic and euglycemic condition.

Dynamic changes in glucose metabolism induced by thiamine deficiency and its replenishment as revealed by a positron autoradiography technique using rat living brain slices

Dynamic changes in the cerebral glucose metabolic rate (CMRglc) before and after thiamine replenishment were investigated in living brain slices obtained from pyrithiamine-treated (PT) and pair-fed control rats by use of a positron autoradiography technique. Fresh rat brain slices (300 microm thick) were incubated with [18F]2-fluoro-2-deoxy-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution at 36 degrees C, during which serial two-dimensional images of [18F]FDG uptake in the slices were constructed on the imaging plates. The net influx constant (=K) of [18F]FDG was determined by a Patlak graphical method of the image data. Prior to thiamine pyrophosphate (TPP)-loading, the K value in the neurologically symptomatic PT was higher in all brain regions except the thalamus and mammillary body than the control, suggesting compensatory enhanced glycolysis. The rapid decrease in this heightened net influx constant immediately after TPP-loading was surmised to be due to activation of pyruvate oxidation with lactate as the substrate, with this inhibiting the glycolysis. From > or = 150 min after TPP-loading, the K value continued to show low values in the thalamus and mammillary body, which are regarded as the responsible sites for Korsakoff syndrome, whereas in all other sites recovery to control values was observed. These findings suggest that using this technique the quantitative evaluation of serial local changes in CMRglc from thiamine deficiency to after its replenishment may be useful in elucidating the pathophysiology and prognosis of Wernickes encephalopathy.

New susceptibility variants to narcolepsy identified in HLA class II region

Narcolepsy, a sleep disorder characterized by excessive daytime sleepiness, cataplexy and rapid eye movement sleep abnormalities, is tightly associated with human leukocyte antigen HLA-DQB1*06:02. DQB1*06:02 is common in the general population (10-30%); therefore, additional genetic factors are needed for the development of narcolepsy. In the present study, HLA-DQB1 in 664 Japanese narcoleptic subjects and 3131 Japanese control subjects was examined to determine whether HLA-DQB1 alleles located in trans of DQB1*06:02 are associated with narcolepsy. The strongest association was with DQB1*06:01 (P = 1.4 × 10(-10), odds ratio, OR = 0.39), as reported in previous studies. Additional predisposing effects of DQB1*03:02 were also found (P = 2.5 × 10(-9), OR = 1.97). A comparison between DQB1*06:02 heterozygous cases and controls revealed dominant protective effects of DQB1*06:01 and DQB1*05:01. In addition, a single-nucleotide polymorphism-based conditional analysis controlling for the effect of HLA-DQB1 was performed to determine whether there were other independent HLA associations outside of HLA-DQB1. This analysis revealed associations at HLA-DPB1 in the HLA class II region (rs3117242, P = 4.1 × 10(-5), OR = 2.45; DPB1*05:01, P = 8.1 × 10(-3), OR = 1.39). These results indicate that complex HLA class II associations contribute to the genetic predisposition to narcolepsy.

Hypoxic tolerance induction in rat brain slices following hypoxic preconditioning due to expression of neuroprotective proteins as revealed by dynamic changes in glucose metabolism

We prepared rat brain slices following sublethal hypoxic pretreatment (preconditioning) and untreated (control) rats, and measured the cerebral glucose metabolic rate (CMRglc) by dynamic positron autoradiography with [18F]2-fluoro-2-deoxy-D-glucose before and after originally lethal 20-min hypoxic loading. In the regions of interest such as the frontal cortex, the CMRglc before hypoxic loading did not differ between the preconditioning and control groups. The CMRglc after reoxygenation was markedly lower than that before hypoxic loading in the control group but did not significantly differ from the preloading value in the preconditioning group. Thus, hypoxic tolerance induction by preconditioning was demonstrated using the maintenance of CMRglc as a neuronal viability index. In addition, profiling of gene expression using an Atlas Rat Stress Array suggested the involvement of the expression of genes such as stress protein in hypoxic tolerance induction.

Effects of chlorpromazine on plasma membrane permeability and fluidity in the rat brain: A dynamic positron autoradiography and fluorescence polarization study

Antipsychotic drugs have been widely used in psychiatry for the treatment of various mental disorders, but the underlying biochemical mechanisms of their actions still remain unclear. Although phenothiazine antipsychotic drugs have been reported to directly interact with the peripheral plasma membrane, it is not known whether these drugs actually affect plasma membrane integrity in the central nervous system. To clarify these issues, we investigated the effect of chlorpromazine (CPZ), a typical phenothiazine antipsychotic drug, on plasma membrane permeability in fresh rat brain slices using a dynamic positron autoradiography technique and [(18)F]2-fluoro-2-deoxy-D-glucose ([(18)F]FDG) as a tracer. Treatment with CPZ (> or =100 microM) resulted in the leakage of [(18)F]FDG-6-phosphate, but not [(18)F]FDG, suggesting that the [(18)F]FDG-6-phosphate efflux was not mediated by glucose transporters, but rather by plasma membrane permeabilization. The leakage of [(18)F]FDG-6-phosphate was followed by slower leakage of cytoplasmic lactate dehydrogenase, suggesting that CPZ could initially induce small membrane holes that enlarged with time. Furthermore, the addition of CPZ (> or =100 microM) caused a decrease in 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy, which implies an increase in membrane fluidity. CPZ loading dose-dependently increased both membrane permeability and membrane fluidity, which suggested the involvement of a perturbation of membrane order in the mechanisms of membrane destabilization induced by antipsychotic drugs.