Koichi Hiraga

Disrupted galectin-3 causes non-alcoholic fatty liver disease in male mice

Galectin‐3, a β‐galactoside‐binding animal lectin, is a multifunctional protein. Previous studies have suggested that galectin‐3 may play an important role in inflammatory responses. Non‐alcoholic fatty liver disease (NAFLD) is increasingly recognized as a liver condition that may progress to end‐stage liver disease and based on the known functions of galectin‐3, it was hypothesized that galectin‐3 might play a role in the development of NAFLD. Thus, this study investigated the role of galectin‐3 in NAFLD by comparing galectin‐3 knockout (gal3−/−) mice and wild‐type (gal3+/+) mice. The livers of gal3−/− male mice at 6 months of age histologically displayed mild to severe fatty change. The liver weight per body weight ratio, serum alanine aminotransferase levels, liver triglyceride levels, and liver lipid peroxide in gal3−/− mice were significantly increased compared with those in gal3+/+ mice. Furthermore, the hepatic protein levels of advanced glycation end‐products (AGE), receptor for AGE (RAGE), and peroxisome proliferator‐activated receptor γ (PPARγ) were increased in gal3−/− mice relative to gal3+/+ mice. In conclusion, this study suggests that the absence of gal3 can cause clinico‐pathological features in male mice similar to those of NAFLD. Copyright

Reduction of the level of the glycine cleavage system in the rat liver resulting from administration of dipropylacetic acid: An experimental approach to hyperglycinemia☆

Abstract Prolonged administration of dipropylacetic acid, a branched-chain fatty acid, reduced the glycine cleavage activity in the liver of rat to about one-third of the activity in the control rat. The reduction of the activity appeared to be due mainly to reduction of the level of P-protein, a pyridoxal phosphate enzyme, which is responsible for the first step of the glycine cleavage, although dipropylacetic acid was also found to inhibit the activity of P-protein in vitro in a noncompetitive but partially competitive manner with respect to glycine. The rat treated with dipropylacetic acid may provide an experimental approach for the biochemical study of hyperglycinemia which accompanies to metabolic disorders of branchedchain keto acids. In the dipropylacetic acid-treated rat, however, the glycine concentration in the serum was not appreciably elevated and this may be accounted for by the fact that the activities of both the glycine cleavage system and serine dehydratase are considerably higher in the rat liver as compared with those in other animals including human.

Glycine cleavage system in ketotic hyperglycinemia: a reduction of H-protein activity.

Glycine cleavage activity was compared in the livers from three cases of ketotic hyperglycinemia (two cases of propionic acidemia and one case of methylmalonic acidemia) and three controls. In one case of propionic acidemia, glycine cleavage activity (5.2 nmole/mg protein/hr) was normal in the liver obtained at biopsy when the patient was well controlled by the treatment with low protein diet (0.8 g/kg/day) and the level of serum glycine was lowered to normal. In the two other cases of ketotic hyperglycinemia, glycine cleavage activity was significantly reduced in the liver obtained at autopsy when the patients died in the state of metabolic acidosis. Its activity in the liver of one case of propionic acidemia (0.7 nmole/mg protein/hr) was 6–26% of that in controls (2.7–10.8 nmole/mg protein/hr), and 2–7% in a case of methylmalonic acidemia (0.2 nmole/mg protein/hr).Analysing of the individual components of the glycine cleavage system, a marked decrease in the activity of H-protein was revealed in the livers of the both patients; it (0.2 nmole/mg protein/ hr) was only 3–4% of that in controls (4.9–6.3 nmole/mg protein/ hr). These findings suggest that the reduction of the glycine cleavage system in the liver of ketotic hyperglycinemia occurs secondarily as speculated previously and is caused mainly by a decrease of H-protein activity.Speculation: The reduction of glycine cleavage activity in ketotic hyperglycinemia may be caused at first by an inactivation of H-protein. Subsequently the activities of other components of the glycine cleavage system may be decreased.

Neuroprotective Role of Transgenic PAF-Acetylhydrolase II in Mouse Models of Focal Cerebral Ischemia

Background and Purpose— Platelet-activating factor (PAF) and oxidized unsaturated free fatty acids have been postulated to aggravate neuronal damage in the postischemic brain. Type II PAF-acetylhydrolase (PAF-AH II) not only terminates signals by PAF by its PAF-hydrolyzing activity but also protects cells against oxidative stress. We examined whether PAF-AH II can rescue cerebral neurons against ischemic insults. Methods— Transgenic mice overexpressing human PAF-AH II in neurons were generated and enzyme expressions were examined biochemically and histochemically. The mice were subjected to 60 minutes of transient middle cerebral artery occlusion followed by reperfusion for 24 hours. The infarction and apoptosis were estimated by TTC staining and fluorescence TUNEL staining, respectively. Results— Overexpression of PAF-AH II was found in brains of transgenic mice by Western blot and enzymatic activity analyses. In immunohistochemistry, human PAF-AH II expression was found throughout the central nervous system, especially in neurons of neocortex, hippocampus, and basal ganglia. The neurological deficit scores, cerebral edema index, and relative infarction volume were all significantly (P<0.05) lower in transgenic mice (1.30±0.72, 1.12±0.04, and 14.0±7.7%, respectively) than in wild-type mice (2.56±0.93, 1.23±0.12, and 31.9±9.7%, respectively). Percentages of apoptotic cells were also significantly (P<0.001) lower in transgenic mice (cortex, 5.2±3.3%; hippocampus, 3.4±7.0%) than in wild-type mice (cortex, 41.1±16.9%; hippocampus, 58.9±15.3%). Conclusions— These results indicate that PAF-AH II exerts strong neuroprotective effects against ischemic injury and suggest a possibility for clinical use of this enzyme in cerebral ischemia.

Nonalcoholic steatohepatitis and hepatocellular carcinoma in galectin‐3 knockout mice

Aim:  Nonalcoholic fatty liver disease (NAFLD) represents a growing health concern due to its rapidly increasing prevalence worldwide. Nonalcoholic steatohepatitis (NASH) is a progressing form of NAFLD, and recently many studies have reported that it could eventually develop into hepatocellular carcinoma (HCC). We previously reported that 6‐month‐old male galectin‐3 knockout (gal3−/−) mice developed clinicopathological features similar to those of NAFLD in humans. Our aim was to investigate the changes in liver histology in gal3−/− mice by long‐term observation.

Contribution of spinal galectin-3 to acute herpetic allodynia in mice.

Summary The present study presents galectin‐3 as a new molecule involved in the spinal processes of acute herpetic pain in mice. Abstract To identify endogenous factors involved in herpetic pain, we performed genome‐wide microarray analysis of the spinal cord of mice that suffered from herpetic allodynia induced by inoculation with herpes simplex virus type 1, which revealed marked induction of galectin‐3, a β‐galactoside‐binding lectin. Therefore, we investigated the role of galectin‐3 in herpetic allodynia. The expression levels of galectin‐3 mRNA and protein were increased with a temporal pattern similar to that of herpetic allodynia. Galectin‐3‐expressing cells were mainly localized in the superficial dorsal horn, round in shape, and positive for the macrophage/microglia markers Iba‐1 and F4/80. In the deep dorsal horn, there were Iba‐1‐positive cells with ramified and stout processes, which were negative for galectin‐3. In the superficial dorsal horn, there were many CD3‐positive T cells, but most of the galectin‐3‐expressing cells were negative for CD3. Galectin‐3‐expressing cells were negative for the neuronal marker NeuN and the astrocyte marker glial fibrillary acidic protein antibody. Deficiency in galectin‐3 markedly reduced herpetic allodynia, without showing an effect on herpes zoster‐like skin lesions. Intrathecal injection of galectin‐3 produced mechanical allodynia in naive mice, and intrathecal injections of anti‐galectin‐3 antibodies significantly reduced herpetic allodynia. The present results suggest that galectin‐3 in infiltrating macrophages and/or resident microglia in the spinal dorsal horn contributes to herpetic allodynia. Galectin‐3 may be a new therapeutic target for the treatment of herpes zoster‐associated pain.

The impaired expression of glycine decarboxylase in patients with hyperglycinemias

Glycine decarboxylase, a constituent of the glycine cleavage system, in patients with either nonketotic or ketotic hyperglycinemia (NKH and KH) was examined using an anti-chicken glycine decarboxylase antibody. Patients with NKH who have lesion in glycine decarboxylase are differentiated by its expressed level in the liver. One group is cases of the neonatal onset type who have neither activity of the enzyme nor protein reactive to the antibody. The other is a case of the late onset type who shows low but detectable activity of the enzyme and the desirable amount of the immunoreactive material. In the liver of a patient with KH not showing the appreciable activity of H-protein, ubiquitous amount of protein reactive to anti-H-protein IgG is detected and amount of glycine decarboxylase has also been lowered. It is suggested that several mechanisms may be involved in determining the expressed level of glycine decarboxylase in patients with hyperglycinemias.

Mutation-, Aging-, and Gene Dosage-dependent Accumulation of Neuroserpin (G392E) in Endoplasmic Reticula and Lysosomes of Neurons in Transgenic Mice

Mutations in human neuroserpin gene cause an autosomal dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB). We generated and analyzed transgenic mice expressing high levels of either FENIB-type (G392E) or wild-type human neuroserpin in neurons of the central nervous system. G392E neuroserpin accumulated age-dependently in neurons of the neocortex, thalamus, amygdala, pons, and spinal cord of homozygous transgenic mice. Such accumulations were not observed in hemizygous transgenic mice nor in transgenic mice for wild-type neuroserpin. In differential centrifugation of brain homogenates, G392E neuroserpin recovered in the nucleus-rich fraction dramatically increased along with aging, suggesting that the aggregations gradually increase their densities presumably by their conversion into heavier and more compact configurations. In immunoelectron microscopical analyses, immunopositivities for G392E neuroserpin were found not only in endoplasmic reticulum but also in lysosomes. G392E neuroserpin transgenic mice were much more susceptible to seizures induced by kainate administration than nontransgenic mice. Overall, G392E neuroserpin accumulated in the central nervous system neurons of transgenic mice in mutation-, aging-, and gene dosage-dependent manners. The established transgenic mice will be valuable to elucidate not only mechanisms for the formation of G392E neuroserpin aggregations but also pathways for the degradation and/or clearance of the already formed aggregations in neurons.

Novel transcript profiling of diffuse alveolar damage induced by hyperoxia exposure in mice: normalization by glyceraldehyde 3-phosphate dehydrogenase.

Under mechanical ventilation with high-inspired oxygen concentration, diffuse alveolar damage was found to take place in some patients. To clarify the molecular pathophysiology of this condition, we investigated the time course of gene expression changes induced by hyperoxia exposure in mouse lung using real-time quantitative polymerase chain reaction (qPCR). Our results normalized by glyceraldehyde 3-phosphate dehydrogenase showed that mRNA levels of cysteine rich protein 61 (CYR61) and connective tissue growth factor (CTGF) were significantly upregulated, while those of surfactant-associated protein C (SFTPC), cytochrome P450, 2F2 (CYP2F2), Claudin 1, (CLDN1), membrane-associated zonula occludens protein-1 (ZO-1), lysozyme (LYZS), and P lysozyme structural (LZP-S) were significantly downregulated. Increasing level of mRNAs, each encoding CYR61 and CTGF, suggests a serious risk of fibrosing alveolitis. Decrease in levels of mRNAs for SFTPC, CYP2F2, CLDN1, ZO-1, LYZS, and LZP-S suggests alveolar dysfunction and disruption of the immune system. Moreover, we confirmed apoptotic conditions, such as significant upregulations of mRNA levels in Myc and Galectin-3. Hyperoxic condition probably yielded reactive oxygen species (ROS), which resulted in a malignant cycle of ROS production by Myc overexpression.

A CELL LINE OF HUMAN MALIGNANT ASTROCYTOMA PRODUCING AUTOCRINE GROWTH FACTOR

SummaryA cell line was established from an anaplastic astrocytoma from a 69-yr-old female. The cells have been serially subcultured over 300 times for 6 yr without showing any sign of cell senescence. Their doubling time is about 36 h. The cells are fusiform and often hexagonal in sparse culture, but become spindle-shaped and formed mosaic structure in confluent culture. Under electron microscopy, intermediate filaments were randomly distributed in the cytoplasma, especially in the perinuclear space. The chromosome number was near tetraploid and varied from 86 to 94 chromosomes with a modal number of 91. The alpha and beta subunits of S-100 protein, vimentin, and glial fibrillary acidic protein (GFAP), which are reliable markers of astrocytic cells, were demonstrated in a large number of cells by immunoperoxidase staining. The results of immunoblotting showed that the expression of vimentin was much higher than that of GFAP. The tumorigenicity of the cells was revealed by xenografting into nude mice, which were X-irradiated before inoculation. Culture medium conditioned by the cells promoted growth of these cells in serum-free conditions and of normal rat glial cells in serum-depleted culture. The growth-promoting effect of conditioned medium was lost by trypsinization and reduced by boiling. These findings suggest that these cells are derived from neoplastic astrocytic cells and secrete a self-acting polypeptide growth-promoting factor into the culture medium.