新井 敏郎

Introduction The prevalence of age-related diseases, including obesity (a lipid metabolism disorder), increases with the increase in a dog’s lifespan. Most of age-related diseases are associated with oxidative stress by excessive production of reactive oxygen species (ROS) from impaired mitochondrial functions. Safe and effective supplements with antioxidative and anti-inflammatory activities are required to prevent obesity and associated complications. Shiitake mushroom exhibit various functions including antioxidant activity. We investigated the effect of shiitake powder supplementation in healthy dogs. Methods Shiitake powder was supplemented at a dose of 800 mg/kg body weight/day for 4 weeks. The dose was set as 0.60–0.65 mg/kg/day of eritadenine, a hypocholesterolemic factor. Results The body weight and body condition score of the dogs did not change after shiitake supplementation. In contrast, plasma total cholesterol concentrations decreased and superoxide dismutase activity and leukocyte sirtuin1 mRNA expression increased significantly in the dogs that received the supplement. Discussion Oral administration of shiitake powder increased antioxidative activity. The supplement may be useful in ameliorating the signs of age-related diseases, including obesity, in dogs.

In older horses, basal metabolic rate decreases, and plasma metabolite and hormone concentrations related to energy metabolism change. The occurrence of age-related diseases, which increases in old animals, may enhance inflammatory reactivity (inflammaging). Finding the appropriate treatment for inflammaging at an early stage may prevent various age-related diseases. Changes in metabolite and hormone concentrations and enzyme activities involved in energy metabolism in the plasma of clinically healthy riding horses of various ages were measured to identify biomarkers of inflammaging (persistent low-grade inflammation that occurs with aging). All horses were clinically healthy, and their body condition scores (BCSs) were 4 or 5 (9-point scale). Plasma triglyceride (TG), total cholesterol (T-Cho), blood urea nitrogen (BUN), insulin concentrations, malondialdehyde (MDA), and serum amyloid A (SAA) concentrations generally increased with age. Adiponectin concentrations, plasma superoxide dismutase (SOD), and leukocyte AMP-activated protein kinase (AMPK) activities decreased, while plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glutathione peroxidase (GPx) remained unchanged as horses aged. Although riding horses that partake in continuous exercise seems to be less likely to develop inflammaging, horses over 17 years of age tend to show proinflammatory signs with disordered lipid metabolism. In riding horses, SAA, in combination with other markers, may be a useful biomarker for inflammaging and dysregulated lipid metabolism in aging horses.

Hospital integration among rural districts to concentrate medical resources is one of the main projects of the Japanese government. In advance of this, we experienced hospital integration and screening for chronic kidney disease as definitive risk for end-stage kidney disease and cardiovascular mortality. After that, high-risk patients have been appropriately referred from generalists to nephrologists and/or cardiologists without acute deterioration of renal function or cardiac sudden death.

H19 is an oncofetal RNA expressed in the developing embryo as well as in bladder, breast, gastric, pancreatic, hepatocellular, and prostate cancers. Recent studies have shown that H19 enhances cancer invasion and metastasis; however, its roles in cancer remain controversial. In the current study, H19 exhibited the second largest increase (82.4-fold) and represented the only non-protein coding gene among 11 genes identified that were elevated over 10-fold in lung-metastasis-derived pancreatic cancer cells compared with their parental cells using a mouse metastatic model. Subsequently, we further clarified the roles of H19 in pancreatic cancer growth and metastasis using in vitro and in vivo techniques. In situ hybridization showed that H19 was detected in 23 of 139 invasive ductal carcinomas (17%), and that H19 expression positively correlated with higher histological grades (P < 0.0001). Overexpression of H19 in PANC-1 pancreatic cancer cells induced higher motilities, whereas H19 inhibition using shRNA and siRNA showed opposite results; however, cell growth rates were not impacted. Intravenous injection of H19 shRNA vector-transfected PANC-1 cells yielded marked inhibition of metastasis in the liver and lungs of immunodeficient mice. These findings suggest that H19 has important roles in pancreatic cancer metastasis, and that inhibition of H19 represents a novel candidate for pancreatic cancer therapy.

As in humans, obesity and its associated diseases represent the most significant threat to the health of veterinary populations. Previous human studies have provided insights into the risk factors of obesity, complex pathogenesis of obesity-associated diseases, and their life-threatening consequences. In humans, the "metabolic syndrome" represents a cluster of metabolic risk factors associated with the development of cardiovascular disease. Risk factors for metabolic syndrome, such as diabetes, obesity, high blood pressure, and its complications increase health-care utilization and medical expenses. Early diagnosis and intervention through preemptive approach is in need, and the new International Diabetes Federation definition of MS serves as the universally accepted diagnostic tool that is accessible in clinical settings. In veterinary populations, especially in cats, similar pathophysiological path and disease progression to the development of MS, such as adipokine dysregulations, chronic inflammation, lipotoxicity, are expected. The aim of this manuscript is twofold. First of all, it presents our preliminary feline obesity study that serves as the basis for discussion of obesity and its metabolic impact on feline population. In this study, we observed the effects of weight gain on energy metabolism using metabolome markers, such as adiponectin (ADN) and proinflammatory cytokines, in correlation with other common biochemical parameters in 14 clinically healthy cats of varying weight status. Further, we evaluated the visceral fat accumulation in three subjects of varying Body Condition Scores via computed tomography imaging and laparoscopic examination, and assessed the adipocyte type and size histologically. Mutually antagonizing changes in ADN and visceral adipose tissue (VAT) reflected the pathophysiological derangements leading to MS earlier than the common biochemical predictors such as glucose, liver values, and lipid markers. Second, it proposes the novel diagnostic and classification method of feline obesity and MS, based on the established diagnostic criteria of human MS and the presented study results. The results supported our novel "classification of feline obesity" and "Feline MS diagnostic criteria," suggesting the need to complement ADN measurement with VAT volume to better understand the pathogenesis of metabolic disturbances in the feline population.

Renal failure is one of the most important social problems for its incurability and high costs for patients' health care. Through clarification of the underlying mechanism for the high susceptibility of cats to renal disease, we here demonstrates that the effective dissociation of serum AIM protein from IgM is necessary for the recovery from acute kidney injury (AKI). In cats, the AIM-IgM binding affinity is 1000-fold higher than that in mice, which is caused by the unique positively-charged amino-acid cluster present in feline AIM. Hence, feline AIM does not dissociate from IgM during AKI, abolishing its translocation into urine. This results in inefficient clearance of lumen-obstructing necrotic cell debris at proximal tubules, thereby impairing AKI recovery. Accordingly, mice whose AIM is replaced by feline AIM exhibit higher mortality by AKI than in wild-type mice. Recombinant AIM administration into the mice improves their renal function and survival. As insufficient recovery from AKI predisposes patients to chronic, end-stage renal disease, feline AIM may be involved crucially in the high mortality of cats due to renal disease. Our findings could be the basis of the development of novel AKI therapies targeting AIM-IgM dissociation, and may support renal function in cats and prolong their lives.

© 2016 The Japanese Society of Veterinary Science. Staphylococcus aureus (SA) is a major cause of bovine mastitis, but its pathogenic mechanism remains poorly understood. To evaluate the role of lipoteichoic acid (LTA) in the immune or inflammatory response of SA mastitis, we investigated the gene expression profile in bovine mammary epithelial cells stimulated with LTA alone or with formalin-killed SA (FKSA) using cap analysis of gene expression. Seven common differentially expressed genes related to immune or inflammatory mediators were up-regulated under both LTA and FKSA stimulations. Three of these genes encode chemokines (IL-8, CXCL6 and CCL2) functioning as chemoattractant molecules for neutrophils and macrophages. These results suggest that the initial inflammatory response of SA infection in mammary gland may be related with LTA induced chemokine genes.

Acarbose (AC) and Sitagliptin (STGP) are oral hypoglycemic agents currently used either alone or in conjunction with human diabetic (Type 2) patients. AC has been used with diabetic cats, but not STGP thus far. Therefore, the objective of this study was to determine the potential use of AC or STGP alone and in combination for diabetic cats, by observing their effect on short-term post-prandial serum glucose, insulin, and incretin hormone (active glucagon-like peptide-1 (GLP-1) and total glucose dependent insulinotropic polypeptide (GIP)) concentrations in five healthy cats, following ingestion of a meal with maltose. All treatments tended (p &lt; 0.10; 5-7.5% reduction) to reduce postprandial glucose area under the curve (AUC), with an accompanying significant reduction (p &lt; 0.05, 35-45%) in postprandial insulin AUC as compared to no treatment. Meanwhile, a significant increase (p &lt; 0.05) in postprandial active GLP-1 AUC was observed with STGP (100% higher) and combined treatment (130% greater), as compared to either AC or no treatment. Lastly, a significant reduction (p &lt; 0.05) in postprandial total GIP AUC was observed with STGP (21% reduction) and combined treatment (7% reduction) as compared to control. Overall, AC, STGP, or combined treatment can significantly induce positive post-prandial changes to insulin and incretin hormone levels of healthy cats. Increasing active GLP-1 and reducing postprandial hyperglycemia appear to be the principal mechanisms of combined treatment. Considering the different, but complementary mechanisms of action by which AC and STGP induce lower glucose and insulin levels, combination therapy with both these agents offers great potential for treating diabetic cats in the future. (C) 2016 Elsevier Ltd. All rights reserved.

The effect of the Fukushima Daiichi Nuclear Power Plant (FNPP) accident on humans and the environment is a global concern. We performed biochemical analyses of plasma from 49 Japanese Black cattle that were euthanized in the ex-evacuation zone set within a 20-km radius of FNPP. Among radionuclides attributable to the FNPP accident, germanium gamma-ray spectrometry detected photopeaks only from Cs-134 and Cs-137 (radiocesium) commonly in the organs and in soil examined. Radioactivity concentration of radiocesium was the highest in skeletal muscles. Assuming that the animal body was composed of only skeletal muscles, the median of internal dose rate from radiocesium was 12.5 mu Gy/day (ranging from 1.6 to 33.9 mu Gy/day). The median of external dose rate calculating from the place the cattle were caught was 18.8 mu Gy/day (6.0-133.4 mu Gy/day). The median of internal and external (total) dose rate of the individual cattle was 26.9 mu Gy/day (9.11-55.1 mu Gy/day). Plasma levels of malondialdehyde and superoxide dismutase activity were positively and glutathione peroxidase activity was negatively correlated with internal dose rate. Plasma alanine transaminase activity and percent activity of lactate dehydrogenase (LDH)-2, LDH-3 and LDH-4 were positively and LDH-1 was negatively correlated with both internal and total dose rate. These suggest that chronic exposure to low-dose rate of ionizing radiation induces slight stress resulting in modified plasma protein and enzyme levels.

Feline mammary carcinomas are characterized by rapid progression and metastases. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis, proliferation and metastasis. The present study aimed to investigate the effects of a single drug therapy of bevacizumab on a xenograft model of feline mammary carcinoma expressing VEGF protein. Bevacizumab treatment suppressed tumor growth by inhibiting angiogenesis and enhancing apoptosis; however, it did not affect the tumor proliferation index. Thus, bevacizumab had anti-tumor effects on a xenograft model, and this may be useful for the treatment of feline mammary carcinoma.

Feline body mass index (fBMI), BW/PCL, length from top of patella to end of calcaneus, was developed as a new diagnostic tool for obesity in cats. To evaluate the effectiveness of fBMI for obese cats in short term, 6 cats were induced weight gain by over-feeding with high fat diet and then they were induced weight reduction by restrict-feeding with low fat diet to measure changes in fBMI and plasma metabolite concentrations and enzyme activities. BCS 3 is correlated with fBMI 24.6-32.0, BCS 4 is correlated with fBMI 33.1-37.1 and BCS 5 is correlated with fBMI 29.9-40.3, respectively. On the correlation coefficient analysis by Pearson's method (P &lt; 0.05), positive correlations (r = 0.403) were seen between the fBMI and plasma triglyceride (TG) levels. From these findings, fBMI seems to be more sensitive and useful indicator for obesity diagnosis in cats.

Calcineurin is a serine/threonine protein phosphatase that consists of catalytic (calcineurin A) and regulatory (calcineurin B) subunits. The conserved protein plays important roles in various biological processes. Drug combination of fluconazole and the calcineurin inhibitor (FK506) showed synergistic effects against dermatophytes. In the current study, we identified the calcineurin A homologous gene (TmcanA) in the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes). Knockdown mutants were produced from A. vanbreuseghemii, resulting in a defection in growth properties in accordance with dose of the suppressing reagent. The TmcanA gene restored the ability of calcineurin A-deficient Cryptococcus neoformans strain to grow at elevated temperatures. Repression of TmcanA at 37 degrees C resulted in severely stunted growth, suggesting that this protein plays a role in tolerance to elevated temperatures. In addition, TMCANA showed an interaction with high osmolarity glycerol (HOG) signalling pathway by governing the secretion of a secondary metabolite. Moreover, expression of the hydrophobin A gene (TmHF) decreased significantly under the TmcanA-repressive condition, suggesting that TMCANA is involved in its regulation. In conclusion, calcineurin A is a multifunctional gene that is involved in the regulation of several biological processes and therefore is worth being considered as a drug target for treatment of dermatophytoses.

Objectives Nestin, a progenitor/stem cell marker, is expressed in human pancreatic cancer, where its expression correlates positively with invasiveness and metastasis. Here, we investigated the inhibition of nestin expression and the regulation of nestin expression. Methods We analyzed the effects of small interfering RNA (siRNA) targeting nestin using pancreatic cancer cell lines. Results Nestin siRNA inhibited the growth, migration, invasion, and sphere-forming ability of the pancreatic cancer cell lines. Pancreatic cancer cells cotreated with gemcitabine and nestin siRNA exhibited lower cell viability than cells treated with a control siRNA, gemcitabine alone, or nestin siRNA alone. Cells derived from the metastatic nodules of mice showed higher nestin expression than the parental cells, and nestin expression in pancreatic cancer cells was regulated by methylation of the nestin gene. In an orthotopic implantation model using mice, administration of nestin siRNA significantly decreased primary and metastatic tumor formation by human pancreatic cancer cells compared to tumor formation in control siRNA-treated mice. Conclusions Nestin plays a key role in pancreatic cancer cell metastasis and stemness and that administration of nestin siRNA may offer a novel therapeutic strategy for pancreatic cancer.

Back ground: Aging is generally associated with alterations in physical activity, weight status and energy metabolism, which predisposes aged individuals to metabolic syndrome. In this manuscript, age effects on energy metabolic indicators of similar physical activity and weight status but of varying ages were investigated. Materials and Methods: Energy metabolic indicators, such as plasma adiponectin, leukocytic AMP-activated protein kinase, plasma malate dehydrogenase and lactate dehydrogenase along with common plasma metabolites, were measured in healthy young (AV = 7.1 years) and aged (AV = 14.1 years) riding horses of similar physical activity, diet and weight status. Malate dehydrogenase and lactate dehydrogenase ratio was also calculated as the indicator of energy metabolism. Results: Plasma adiponectin concentration and leukocytic AMP-activated protein kinase activity in aged horses were significantly lower than those in young horses (p&lt 0.05, Mann-Whitney U test). Although not significant, energy metabolism indicators, malate dehydrogenase, lactate dehydrogenase and their ratio were lower in aged group when compared to those of young group. Conclusion: The present results indicate the decline in energy metabolism with aging in healthy horses even without any visible changes in adiposity. Such changes reflect dysfunction of energy metabolism and predispose the aged individuals to the development of metabolic syndrome.

Many veterinarians feel that feline obesity is increasing in prevalence within Japan, but are unsure as to what real rate of obesity prevalence exists currently. The objectives of this study are twofold. First, we sought to determine the ratio of overweight/obese cats, as determined via body condition score (BCS), based on a cross-sectional pool of healthy cats. Second, using a uniform method of biochemical measurement for cat plasma collected all over Japan, we sought to construct cross-sectional working reference ranges of plasma metabolites using age, BCS, and sex as classifying factors. Overall, the rate of overweight and obese cats (BCS of &gt;3) was 56%, whereas obese cats (BCS &gt;4) constituted 42% of our pool. Aging leads to an increased risk of developing lipid metabolism and kidney issues. Increases in BCS and age both carry increased risk of insulin resistance development. Moreover, increases in BCS bring about a higher risk of developing lipid metabolism problems, which may lead to the development of high cholesterol and triglyceride lipidemia, accompanied by higher levels of free fatty acids. Males appear to have a higher risk than females of developing higher BCS and body weight values, in addition to higher insulin and lower adiponectin values.

In dogs, hyperadrenocorticism (HAC) is associated with insulin resistance and diabetes does progress with HAC. There are significant differences in the transcriptomic and proteomic patterns of activated T cells, which parallel the findings in muscle tissues. The aim of this study was to assess how glucocorticoids affect intracellular metabolites in canine peripheral blood mononuclear cells (CnPBMCs) using dexamethasone. A total of 96 metabolites were identified by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). After incubation with dexamethasone, the metabolites glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, sedoheptulose 7-phosphate and acetyl-CoA were significantly increased. However, ATP, CTP, dATP, pyruvic acid and NADP(+) were significantly decreased. These results show that a glucocorticoid reduces the catabolic reaction of glucose and accordingly decreases the glucose requirements of CnPBMCs. (C) 2015 Elsevier Ltd. All rights reserved.

Nuclear factor xB (NF-kappa B) is a key factor in the development of chronic inflammation and is deeply involved in age-related and metabolic diseases development. These diseases have become a serious problem in cats. Sirtuin 1 (SIRT1) is associated with aging and metabolism through maintaining inflammation via NF-kappa B. In addition, fibroblasts are considered an important factor in the development of chronic inflammation. Therefore, we aimed to examine the effect of cat SIRTI (cSIRT1) on NF-kappa B in cat fibroblast cells. The up-regulation of NF-kappa B transcriptional activity and pro-inflammatory cytokine mRNA expression by p65 subunit of NF-kappa B and lipopolysaccharide was suppressed by cSIRT1 in cat fibroblast cells. Our findings show that cSIRT I is involved in the suppression of inflammation in cat fibroblast cells.

Oxidized low-density lipoprotein (LDL) is thought to play an important role in the inflammatory response associated with human obesity. The purpose of this preliminary study was to determine oxidized LDL concentrations in healthy dogs and cats, and to evaluate whether obesity affects oxidized LDL concentration, using 39 cats and 19 dogs that had visited two different veterinary clinics in Japan. We hypothesized that oxidized LDL concentrations measured against body condition score (BCS) may have a potential value in evaluating the qualities of accumulated or circulating lipids in obese dogs and cats that do not show signs of metabolic diseases. The mean oxidized LDL value in BCS3 dogs (2.4 ± 0.9 μg/dl) was very similar to that of BCS5 dogs (2.2 ± 0.3 μg/dl). The mean oxidized LDL value of BCS4 dogs was 7.2 ± 10.3 μg/dl and the highest among three groups. BCS4 dogs included two dogs whose oxidized LDL values were higher than the mean oxidized LDL value of healthy humans (11.2 ± 0.3 μg/dl). On the other hand, the mean oxidized LDL value of BCS3 cats was 2.5 ± 0.9 μg/dl, and those of BCS4 and 5 cats were higher than that of BCS3, but there was no significant difference. The BCS4 cat group included one cat with a higher oxidized LDL value, and the BCS5 group also included two cats with oxidized LDL values higher than the mean oxidized LDL value of healthy humans. Interestingly, the oxidized LDL values in two obese dogs and three obese cats were indeed higher than the mean oxidized LDL value of humans with coronary artery disease (20.1 ± 1.1 μg/dl). In conclusion, this preliminary study showed reference ranges of oxidized dogs and cats against BCS. Obesity alone does not appear to have any direct effect on serum oxidized LDL values in healthy dogs and cats.

Background: Obesity and overweight have been frequently observed in dogs and cats in recent years as in humans. The compositions of fatty acids (FAs) in the accumulated lipids in tissues of obese animals may have important roles in the process and mechanisms related to the onset of metabolic disorders. The purpose of this study was to evaluate the effects of a high fat (HF) diet, which contained a higher proportion of saturated FAs, on FA metabolism and distribution in obese cats. Cats (N = 12) were divided into control diet group (crude fat; 16.0 %) (n = 4) or a high fat (HF) diet group (crude fat; 23.9 %) (n = 8). The HF diet contained up to 60 % of calories from fat and was rich in stearic acid. Blood samples were collected at 0, 2, 4 and 6 weeks after the feeding. Adipose and liver tissues were collected at the 6th week after feeding. We performed analysis of histological findings and fatty acid composition in serum and tissues. Results: Body weights of the cats significantly increased in the HF group. The increased activities of hepatic enzymes and the accumulation of lipid droplets were found in hepatocytes in the HF group at the 6th week after feeding. In this study, the stearic acid (C18:0)-rich HF diet contained less oleic acid (C18:1n-9) and more linoleic acid (C18:2n-6) than the control. However, the composition of oleic acid in the liver was higher, and those of stearic acid and linoleic acid were lower in the HF group at the 6th week after feeding. The higher oleic acid: stearic acid ratio suggests an increase in the conversion from saturated FA to mono-unsaturated FAs, which may reflect the hepatic storage of FAs as a relatively harmless form. Conclusion: The stearic acid-rich HF diet increased hepatic lipid accumulation accompanied by the increased of hepatic oleic acid, increased serum oleic acid and activation of hepatic enzymes. These findings could be an important sign of early stages of dyslipidemia and hepatic damage. Also, the higher oleic acid: stearic acid ratio might be related to the increased activity of SCD-1, which suggests that the stearic acid-rich HF diet evoked hepatic lipogenesis in the feline liver.

Nuclear factor kappa B (NF-kappa B) plays an important role in the immune system. The p65 subunit is an important part of NF-kappa B unit, and studies of dog and cat p65 subunits of NF-kappa B (dp65 and cp65) are important in understanding their immune function. In this study, we described the molecular characterization of dp65 and cp65. The dp65 and cp65 complementary DNA encoded 542 and 555 amino acids, respectively, showing a high sequence homology with the mammalian p65 subunit (&gt;87.5%). Quantitative polymerase chain reaction revealed that the p65 messenger RNA is highly expressed in the dog stomach and cat heart and adipose tissue. Functional NF-kappa B promoter-luciferase reporter vectors revealed that our isolated dp65 and cp65 cDNA encodes a functionally active protein. Transiently expressed dp65 and cp65 up-regulated pro-inflammatory cytokine expression levels in dog and cat, respectively. These findings suggest that dp65 and cp65 play important roles in regulating immune function. (C) 2015 Elsevier Ltd. All rights reserved.

Nestin, a class VI intermediate filament, was first described as a neuronal stem/progenitor cell marker. We previously reported that knockdown of nestin expression in human glioblastoma cells suppresses cell proliferation, migration, and invasion. In the present study, we examined the effect of nestin on sternness, and identified molecules involved in modulating nestin function in glioblastoma cells. Nestin expression was shown to be higher in high-grade gliomas than in low-grade gliomas. Furthermore, compared with control cells, nestin short hairpin RNA (shRNA)-transfected glioblastoma cells exhibited reduced sphere formation, decreased expression of NANOG, N-cadherin, CD133, and Oct-4, and decreased tumor size in vivo. To examine the proteins regulated by nestin in glioblastomas, we carried out two-dimensional electrophoresis using nestin shRNA-transfected glioblastoma cells. As a result, nestin shRNA-transfected glioblastoma cells exhibited a decrease in the level of phosphorylation of heat shock cognate 71 kDa protein (HSC71; gene HSPA8). From immunoprecipitation experiments, we demonstrated the direct binding of nestin, HSC71, and cyclin D1 in vitro. Overexpression of nestin in glioblastoma cells increased cell growth, sphere formation, and cell invasion. Transfection with HSC71 siRNA restored nestin expression and cell behavior; therefore, HSC71 knockdown will interfere with enhanced tumorigenic properties of glioblastoma cells that ectopically overexpress nestin. We have demonstrated that HSC71 and nestin regulate each other's expression levels or patterns, and that cyclin D1 is located downstream of nestin and HSC71. In conclusion, nestin regulates sternness, cell growth, and invasion in glioblastoma cells through the alteration of HSC71. Inhibition of nestin and HSC71 may thus be a useful molecular target in the treatment of glioblastomas. (C) 2014 Elsevier Ireland Ltd. All rights reserved.