Katsura Mizushima

The microRNA miR-696 regulates PGC-1α in mouse skeletal muscle in response to physical activity

MicroRNAs (miRNAs) are small noncoding RNAs involved in posttranscriptional gene regulation that have been shown to be involved in growth, development, function, and stress responses of various organs. The purpose of this study was to identify the miRNA response to physical activity, which was related to functions such as nutrient metabolism, although the miRNAs involved are currently unknown. C57BL/6 mice were divided into exercise and control groups. The exercise group performed running exercise, with a gradual increase of the load over 4 wk. On the other hand, to examine the effect of muscle inactivity, the unilateral hindlimbs of other mice were fixed in a cast for 5 days. Microarray analysis for miRNA in gastrocnemius revealed that miR-696 was markedly affected by both exercise and immobilization, showing opposite responses to these two interventions. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), which was increased by exercise and decreased by immobilization in the protein level, was predicted as a target regulated by miR-696. In cultured myocytes, intracellular miR-696 variation led to negative regulation of PGC-1alpha protein along with the expression of mRNAs for downstream genes. In addition, we found decreases in the biogenesis of mitochondria and fatty acid oxidation in miR-696-overexpressing myocytes compared with normal control myocytes. These observations demonstrate that miR-696 is a physical activity-dependent miRNA involved in the translational regulation of PGC-1alpha and skeletal muscle metabolism in mice.

Increased expression of microRNA in the inflamed colonic mucosa of patients with active ulcerative colitis

Background and Aims:  MicroRNA (miRNA) are endogenous, approximately 22‐nucleotide non‐coding RNA that suppress gene expression at post‐transcriptional levels by binding to the 3′‐untranslated region of specific mRNA targets through base‐pairing. It has been recently reported that miRNA have critical functions in key biological processes such as cell proliferation and cell death in various cancer cells. However, the relationship between intestinal inflammation and miRNA expression remains unclear. In the present study, we used microarray technology to identify miRNA induced in the colonic mucosa of patients with active ulcerative colitis (UC).

A novel myokine, secreted protein acidic and rich in cysteine (SPARC), suppresses colon tumorigenesis via regular exercise

Objective Several epidemiological studies have shown that regular exercise can prevent the onset of colon cancer, although the underlying mechanism is unclear. Myokines are secreted skeletal muscle proteins responsible for some exercise-induced health benefits including metabolic improvement and anti-inflammatory effects in organs. The purpose of this study was to identify new myokines that contribute to the prevention of colon tumorigenesis. Methods To identify novel secreted muscle-derived proteins, DNA microarrays were used to compare the transcriptome of muscle tissue in sedentary and exercised young and old mice. The level of circulating secreted protein acidic and rich in cysteine (SPARC) was measured in mice and humans that performed a single bout of exercise. The effect of SPARC on colon tumorigenesis was examined using SPARC-null mice. The secretion and function of SPARC was examined in culture experiments. Results A single bout of exercise increased the expression and secretion of SPARC in skeletal muscle in both mice and humans. In addition, in an azoxymethane-induced colon cancer mouse model, regular low-intensity exercise significantly reduced the formation of aberrant crypt foci in wild-type mice but not in SPARC-null mice. Furthermore, regular exercise enhanced apoptosis in colon mucosal cells and increased the cleaved forms of caspase-3 and caspase-8 in wild-type mice but not in SPARC-null mice. Culture experiments showed that SPARC secretion from myocytes was induced by cyclic stretch and inhibited proliferation with apoptotic effect of colon cancer cells. Conclusions These findings suggest that exercise stimulates SPARC secretion from muscle tissues and that SPARC inhibits colon tumorigenesis by increasing apoptosis.

Muscle-enriched microRNA miR-486 decreases in circulation in response to exercise in young men

Background: MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation. miRNAs are taken in by intracellular exosomes, secreted into circulation, and taken up by other cells, where they regulate cellular functions. We hypothesized that muscle-enriched miRNAs existing in circulation mediate beneficial metabolic responses induced by exercise. To test this hypothesis, we measured changes in muscle-enriched circulating miRNAs (c-miRNAs) in response to acute and chronic aerobic exercise. Methods: Eleven healthy young men (age, 21.5 ± 4.5 y; height, 168.6 ± 5.3 cm; and body weight, 62.5 ± 9.0 kg) performed a single bout of steady-state cycling exercise at 70% VO2max for 60 min (acute exercise) and cycling training 3 days per week for 4 weeks (chronic exercise). Blood samples were collected from the antecubital vein before and after acute and chronic exercise. RNA was extracted from serum, and the levels of muscle-enriched miRNAs (miR-1, miR-133a, miR-133b, miR-206, miR-208b, miR-486, and miR-499) were measured. Results: All of these miRNAs, except for miR-486, were found at too low copy numbers at baseline to be compared. miR-486 was significantly decreased by both acute (P = 0.013) and chronic exercise (P = 0.014). In addition, the change ratio of miR-486 due to acute exercise showed a significant negative correlation with VO2max for each subject (R = 0.58, P = 0.038). Conclusion: The reduction in circulating miR-486 may be associated with metabolic changes during exercise and adaptation induced by training.

Astaxanthin Protects Mesangial Cells From Hyperglycemia-Induced Oxidative Signaling

Astaxanthin (ASX) is a carotenoid that has potent protective effects on diabetic nephropathy in mice model of type 2 diabetes. In this study, we investigated the protective mechanism of ASX on the progression of diabetic nephropathy using an in vitro model of hyperglycemia, focusing on mesangial cells. Normal human mesangial cells (NHMCs) were cultured in the medium containing normal (5 mM) or high (25 mM) concentrations of D‐glucose. Reactive oxygen species (ROS) production, the activation of nuclear transcription factors such as nuclear factor kappa B (NFκB) and activator protein‐1 (AP‐1), and the expression/production of transforming growth factor‐beta 1 (TGFβ1) and monocyte chemoattractant protein‐1 (MCP‐1) were evaluated in the presence or absence of ASX. High glucose (HG) exposure induced significant ROS production in mitochondria of NHMCs, which resulted in the activation of transcription factors, and subsequent expression/production of cytokines that plays an important role in the mesangial expansion, an important event in the pathogenesis of diabetic nephropathy. ASX significantly suppressed HG‐induced ROS production, the activation of transcription factors, and cytokine expression/production by NHMCs. In addition, ASX accumulated in the mitochondria of NHMCs and reduced the production of ROS‐modified proteins in mitochondria. ASX may prevent the progression of diabetic nephropathy mainly through ROS scavenging effect in mitochondria of mesangial cells and thus is expected to be very useful for the prevention of diabetic nephropathy. J. Cell. Biochem. 103: 1925–1937, 2007.

A Mouse Model of Metabolic Syndrome; Increase in Visceral Adipose Tissue Precedes the Development of Fatty Liver and Insulin Resistance in High-Fat Diet-Fed Male KK/Ta Mice.

To determine the relative contribution of obesity and visceral white adipose tissue (WAT) to metabolic syndrome, we developed a model that is susceptible to high-fat diet-induced obesity and insulin resistance using male KK/Ta mice. The ratio of WAT weight to body weight was greater in the high-fat diet group compared with the control group in 10-, 14-, and 22-week-old mice. The increase in visceral WAT preceded development of fatty liver and insulin resistance. Adiponectin mRNA expression in WAT was markedly decreased before the decrease in its plasma levels or the development of insulin resistance. Insulin resistance appeared in association with fatty infiltration and TNF-α expression in the liver in 22-week-old mice. These data indicate that our mouse model would be useful for future studies that investigate the role of visceral WAT and its products in the development of metabolic syndrome.

Reactive oxygen species-quenching and anti-apoptotic effect of polaprezinc on indomethacin-induced small intestinal epithelial cell injury

BackgroundTo protect the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs is one of the critical issues in the field of gastroenterology. Polaprezinc (PZ), a gastric muco-protecting agent, has been widely used for the treatment of gastric ulcer and gastritis for its unique effects, such as its strong reactive oxygen species (ROS)-quenching effect. The aim of this study was to clarify the mechanism by which indomethacin-induced small intestinal mucosal injury occurs, by using a rat intestinal epithelial cell line (RIE-1). In addition, the protective role of PZ and the possible mechanism of its effect on indomethacin-induced small intestinal injury were investigated.MethodsCell death was evaluated by methyl thiazolyl tetrazolium (MTT) assay and a double-staining method with Hoechst33342 dye and propidium iodide. Indomethacin-induced ROS production was evaluated by detecting the oxidation of a redox-sensitive fluorogenic probe, RedoxSensor, and the oxidation of cysteine residues of proteins (protein S oxidation). The activation of cytochrome c, smac/DIABLO, and caspase-3 was assessed by western blotting. In some experiments, PZ or its components, l-carnosine and zinc, were used.ResultsWe found that indomethacin caused apoptosis in RIE-1 cells in a dose- and time-dependent manner. Indomethacin also induced ROS production and an increase in the protein S oxidation of RIE-1. Pretreatment of RIE-1 with PZ or zinc sulfate, but not l-carnosine, significantly reduced the indomethacin-induced apoptosis. PZ prevented ROS production and the increase in protein S-oxidation. PZ inhibited indomethacin-induced cytochrome c and smac/DIABLO release and subsequent caspase-3 activation.ConclusionsThe protective effect of PZ on indomethacin-induced small intestinal injury may be dependent on its ROS-quenching effect.

Lansoprazole, a Proton Pump Inhibitor, Mediates Anti-Inflammatory Effect in Gastric Mucosal Cells through the Induction of Heme Oxygenase-1 via Activation of NF-E2-Related Factor 2 and Oxidation of Kelch-Like ECH-Associating Protein 1

Induction of heme oxygenase-1 (HO-1) expression has been associated with cytoprotective and anti-inflammatory actions of lansoprazole, a proton pump inhibitor, but the underlying molecular mechanisms remain largely unresolved. In this study, we investigate the role of transcriptional NF-E2-related factor 2 (Nrf2), its phosphorylation/activation, and oxidation of Kelch-like ECH-associating protein 1 (Keap1) in lansoprazole-induced HO-1 up-regulation using cultured gastric epithelial cells (rat gastric mucosal cell line, RGM-1). HO-1 expression of RGM-1 cells was markedly enhanced in a time- and dose-dependent manner by the treatment with lansoprazole, and this up-regulation of HO-1 contributed to the inhibition of chemokine production from stimulated RGM-1 cells. Transfection of Nrf2-siRNA suppressed the lansoprazole-induced HO-1. An electrophoretic mobility shift assay showed increases in the nuclear translocation and stress-response elements (StRE) binding activity of Nrf2 proteins in RGM-1 cells treated with lansoprazole. Furthermore, in RGM-1 cells transfected with HO-1 enhancer luciferase reporter plasmid containing mutant StRE, lansoprazole-induced HO-1 reporter gene activity was diminished. Lansoprazole promoted the phosphorylation of extracellular signal-regulated kinase (ERK), and lansoprazole-induced HO-1 up-regulation was suppressed by U0126, an ERK-specific inhibitor. Phosphorylated Nrf2 protein was detected in the phosphoprotein fraction purified by a Pro-Q Diamond Phosphoprotein Enrichment kit. Finally, an oxidative form of the Keap1 protein was detected in lansoprazole-treated RGM-1 cells by analyzing S-oxidized proteins using biotinylated cysteine as a molecular probe. These results indicate that lansoprazole up-regulates HO-1 expression in rat gastric epithelial cells, and the up-regulated HO-1 contributes to the anti-inflammatory effects of the drug. Phosphorylation of ERK and Nrf2, activation and nuclear translocation of Nrf2, and oxidation of Keap1 are all involved in the lansoprazole-induced HO-1 up-regulation.

Carbon Monoxide Liberated from Carbon Monoxide-Releasing Molecule Exerts an Anti-inflammatory Effect on Dextran Sulfate Sodium-Induced Colitis in Mice

BackgroundEndogenous carbon monoxide (CO) is one of the three products of heme degradation by heme oxygenase-1 (HO-1) and exerts novel anti-inflammatory and anti-apoptotic effects as a gaseous second messenger. The purpose of this investigation was to determine whether exogenous CO could modulate intestinal inflammation.MethodsAcute colitis was induced with 2% DSS in male C57BL/6 mice. CO-releasing molecule-2 (CORM-2; tricarbonyldichlororuthenium(II) dimer) was intraperitoneally administered twice daily and the disease activity index (DAI) was determined. We measured tissue-associated myeloperoxidase (MPO) activity as an index of neutrophil infiltration, and the production of keratinocyte chemoattractant (KC) and tumor necrosis factor-α (TNF-α) protein in the intestinal mucosa. In an in-vitro study, young adult mouse colonic epithelial (YAMC) cells were incubated with TNF-α, and KC mRNA/protein expression and nuclear translocation of nuclear factor-kappa B (NF-κB) were measured with or without CORM-2 treatment.ResultsAfter DSS administration, DAI score increased in a time-dependent manner, and this increase was ameliorated by CORM-2 treatment. Increases in MPO activity and in the production of KC and TNF-α after DSS administration were significantly inhibited by CORM-2. TNF-α-induced KC production in YAMC cells was also inhibited by CORM-2 treatment. Further, nuclear translocation of NF-κB in YAMC cells was inhibited by CORM-2.ConclusionCORM-liberated CO significantly inhibited inflammatory response in murine colitis by inhibition of cytokine production in the colonic epithelium. These results suggest that CO could become a new therapeutic molecule for inflammatory bowel disease.

Whole body hyperthermia improves obesity-induced insulin resistance in diabetic mice

Aim: In this study, we examined the efficacy of whole body hyperthermia (WBH) on obesity-induced insulin resistance in diabetic mice. Methods: Male db/db mice were treated with WBH 3 times per week for 12 weeks. The rectal temperature of mice reached 38.0°C 5 min after heating, and was kept at 38.0°C for 30 min. At the end of each week, tail snip glucose levels were determined under fasting conditions. The GLUT-4 gene expression of muscle tissue was analyzed by real-time PCR. Results: (1) WBH-treated db/db mice showed a significant decrease in fasting blood glucose level as compared with untreated db/db mice (p < 0.01). (2) Plasma insulin levels in untreated db/db mice at the age of 10 weeks were significantly increased compared with those of db/+ mice (p < 0.0001). On the other hand, the reduction (31%) in insulin levels in WBH-treated mice indicated improved insulin sensitivity. (3) The ability of WBH to increase insulin sensitivity was further established in glucose tolerance tests and insulin tolerance tests. (4) Urine albumin of db/db mice significantly increased compared with those of db/+ mice at 18 weeks of age (p < 0.001). This increase in urinary albumin was significantly inhibited by WBH (p < 0.01). (5) WBH up-regulated the expression of GLUT4 mRNA in skeletal muscle. Conclusion: Although the mechanisms have not yet been completely investigated, WBH may provide a new therapeutic or preventive modality against obesity-related diseases such as T2DM and metabolic or insulin resistance syndrome.