Toshiro Arai

Comparison of bone marrow and adipose tissue-derived canine mesenchymal stem cells

BackgroundBone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) are potential cellular sources of therapeutic stem cells. MSCs are a multipotent population of cells capable of differentiating into a number of mesodermal lineages. Treatment using MSCs appears to be a helpful approach for structural restoration in regenerative medicine. Correct identification of these cells is necessary, but there is inadequate information on the MSC profile of cell surface markers and mRNA expression in dogs. In this study, we performed molecular characterization of canine BM-MSCs and AT-MSCs using immunological and mRNA expression analysis.ResultsSamples were confirmed to be multipotent based on their osteogenic and adipogenic differentiation. And these cells were checked as stem cell, hematopoietic and embryonic stem cell (ESC) markers by flow cytometry. BM- and AT-MSCs showed high expression of CD29 and CD44, moderate expression of CD90, and were negative for CD34, CD45, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. SSEA-1 was expressed at very low levels in AT-MSCs. Quantitative real-time PCR (qRT-PCR) revealed expression of Oct3/4, Sox2, and Nanog in BM- and AT-MSCs. There was no significant difference in expression of Oct3/4 and Sox2 between BM-MSCs and AT-MSCs. However, Nanog expression was 2.5-fold higher in AT-MSCs than in BM-MSCs. Using immunocytochemical analysis, Oct3/4 and Sox2 proteins were observed in BM- and AT-MSCs.ConclusionOur results provide fundamental information to enable for more reproducible and reliable quality control in the identification of canine BM-MSCs and AT-MSCs by protein and mRNA expression analysis.

Comparison of expression of glucokinase gene and activities of enzymes related to glucose metabolism in livers between dog and cat.

Plasma glucose and immunoreactive insulin (IRI) concentrations and activities of enzymes related to glucose metabolism in livers were measured in dogs and cats. Nucleotide sequences of the conserved region of glucokinase (GK) cDNA that contained ATP- and glucose-binding domains were determined in canine liver and feline pancreas for design of the species-specific oligonucleotide primers for reverse transcription–polymerase chain reaction (RT-PCR) analysis. There were no significant differences in plasma glucose and IRI concentrations between dogs and cats. In feline liver, although GK activities were not detected, activities of hexokinase, fructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, fructose-1,6-bisphosphatase and glucose-6-phosphatase were significantly higher than those in canine liver. The partial sequences of canine liver GK and feline pancreas GK cDNA were respectively 88% and 89% identical with the rat liver GK cDNA. Expression of GK gene was observed in canine liver and pancreas and feline pancreas with RT-PCR using species specific primers based on the cDNA sequences.

Hepatic enzyme activities and plasma insulin concentrations in diabetic herbivorous voles

The activities of the hepatic glycolytic enzymes glucokinase (GKase) and hexokinase (HKase) in herbivorous Microtus arvalis were very low and the hepatic fructose-1,6-diphosphatase (FDPase) activities were aomost the same as those in C57BL/6J mice. Glycosuria was observed in over 50% of voles fed on a low fibre, high concentrate diet. Voles with a high incidence of glycosuria for over 6 weeks became insulin deficient. In these diabetic voles, the hepatic GKase, HKase and FDPase activities decreased considerably as a result of diminished insulin secretion and fatty degeneration of the hepatic cells. It was considered that M. arvalis would be a useful animal model in which to study disorders of glucose utilization in herbivora.

d-Glucose transport activities in erythrocytes and hepatocytes of dogs, cats and cattle

1. The activities of D-glucose transport and hexokinase were investigated in erythrocytes or hepatocytes of dogs, cats and cattle. 2. The mean D-glucose transport activity in erythrocytes of dogs was 6.0 nmol/min/mg protein, half the value of hepatocytes. 3. The activities of D-glucose transport in erythrocytes and hepatocytes or hepatic hexokinase of cats were about one-third of those of dogs. 4. Cattle with low blood glucose concentrations showed considerably low activities of D-glucose transport and hexokinase, about one-third of those of dogs.

Predisposition for primary hyperlipidemia in Miniature Schnauzers and Shetland sheepdogs as compared to other canine breeds.

Miniature Schnauzers are the first canine breed, in the United States, reported to suffer from primary hyperlipidemia, but this has yet to be documented in other regions. Using over 900 canine plasma samples collected from over seven different veterinary clinics across Japan, the aim of this study was to compare plasma triglyceride (TG) and cholesterol concentrations between Miniature Schnauzers and other purebreeds in Japan. In addition, we investigated the influence of aging and sex on changes to hyperlipidemia incidence in purebred dogs. Our results indicated that both Miniature Schnauzers and Shetland sheepdogs in Japan exhibited remarkably high concentrations of plasma TG and total cholesterol, which are considered to be signs of hyperlipidemia, as compared to other purebred and mixed (Mongrel) canine breeds. Interestingly, the cause and conditions of primary hyperlipidemia in Miniature Schnauzers and Shetland sheepdogs might be different, with hypertriglyceridemia predominantly occurring with Miniature Schnauzers and hypercholesterolemia occurring in Shetland sheepdogs. However, with the influence of aging, the hyperlipidemia evolves into both hypercholesterolemia and hypertriglyceridemia in both groups indicating that the severity of hyperlipidemia positively correlates with aging. Gender differences were also observed with regards to severity. In fact, a higher severity was prevalent with female Miniature Schnauzers than their male counterparts whereas it was more balanced between genders for Shetland sheepdogs.

Cerebrospinal fluid biomarkers showing neurodegeneration in dogs with GM1 gangliosidosis : possible use for assessment of a therapeutic regimen

The present study investigated cerebrospinal fluid (CSF) biomarkers for estimating degeneration of the central nervous system (CNS) in experimental dogs with GM1 gangliosidosis and preliminarily evaluated the efficacy of long-term glucocorticoid therapy for GM1 gangliosidosis using the biomarkers identified here. GM1 gangliosidosis, a lysosomal storage disease that affects the brain and multiple systemic organs, is due to an autosomal recessively inherited deficiency of acid beta-galactosidase activity. Pathogenesis of GM1 gangliosidosis may include neuronal apoptosis and abnormal axoplasmic transport and inflammatory response, which are perhaps consequent to massive neuronal storage of GM1 ganglioside. In the present study, we assessed some possible CSF biomarkers, such as GM1 ganglioside, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), neuron-specific enolase (NSE) and myelin basic protein (MBP). Periodic studies demonstrated that GM1 ganglioside concentration, activities of AST and LDH, and concentrations of NSE and MBP in CSF were significantly higher in dogs with GM1 gangliosidosis than those in control dogs, and their changes were well related with the months of age and clinical course. In conclusion, GM1 ganglioside, AST, LDH, NSE and MBP could be utilized as CSF biomarkers showing CNS degeneration in dogs with GM1 gangliosidosis to evaluate the efficacy of novel therapies proposed for this disease. In addition, we preliminarily treated an affected dog with long-term oral administration of prednisolone and evaluated the efficacy of this therapeutic trial using CSF biomarkers determined in the present study. However, this treatment did not change either the clinical course or the CSF biomarkers of the affected dog, suggesting that glucocorticoid therapy would not be effective for treating GM1 gangliosidosis.

Activities of enzymes in the malate-aspartate shuttle and the isoenzyme pattern of lactate dehydrogenase in plasma and peripheral leukocytes of lactating Holstein cows and riding horses.

The activities of the enzymes involved in the malate-aspartate shuttle and lactate dehydrogenase (LDH) and the pattern of the isoenzymes of LDH were determined in plasma and peripheral leukocytes of lactating Holstein cows and thoroughbred riding horses as representative herbivorous animals. In the horse plasma, LDH activities were significantly lower and AST activities were significantly higher than those in the cow plasma. The specific activities of cytosolic malate dehydrogenase (MDH), LDH and AST in the horse leukocytes were higher than those in the cows. The cytosolic ratio of MDH/LDH activity (ML ratio) in the horse leukocytes was significantly lower than that in the cow leukocytes owing to significantly higher activities of LDH. The ML ratio was considered to reflect the difference in energy metabolism in leukocytes between cows and horses. The plasma LDH isoenzyme patterns of cow and horse showed the characteristic as herbivorous animals with dominance of LDH-1, -2 and -3. The LDH isoenzyme patterns with dominance of LDH-3 and -4 in the horse leukocytes were remarkably different from those in the cow leukocytes. There were significant differences in activities of malate-aspartate shuttle enzymes, ML ratio and LDH isoenzyme patterns in the cytosolic fractions of leukocytes between the lactating cows and the riding horses.

Decreased gene expression of insulin signaling genes in insulin sensitive tissues of obese cats

Type 2 diabetes mellitus (DM) animal models have provided ample opportunity for investigating pathogenesis, as well as to evaluate novel treatment and prevention options for the disease. Because the domestic cat shares a similar environment with humans, it is also confronted with many similar risk factors for diabetes, such as physical inactivity and obesity. Obesity is a significant risk factor for diabetes in cats, and as such, the domestic cat may serve as an ideal model for investigating obesity induced insulin resistance. This study determined changes in insulin signaling genes within insulin sensitive tissues of obese felines. Quantitative RT-PCR was performed to determine mRNA levels of three important insulin signaling genes which have been implicated with insulin resistance: insulin receptor substrate (IRS)-1, IRS-2, and phosphatidylinositol 3’-kinase (PI3-K) p85α. Obese cats had significantly lower IRS-2 and PI3-K p85α mRNA levels in liver and skeletal muscle as compared to control cats. This down regulation of insulin signaling genes in obese cats mirrors that of obese humans and rodents suffering from insulin resistance. Interestingly, preprandial blood tests indicated that our obese cats were no different from control cats with regards to glucose tolerance and insulin resistance, thus indicating that the obese cats used in our study had a moderate level of obesity. Therefore, insulin signaling gene alterations were occurring in insulin sensitive tissues of moderately obese felines before glucose intolerance was clinically evident. As such, the monitoring of key insulin signaling genes may have some important diagnostic value to determine the risk level and degree of obesity induced insulin resistance.

Obesity induced changes to plasma adiponectin concentration and cholesterol lipoprotein composition profile in cats.

Feline obesity generally results in aberrations to plasma metabolite levels, such as lipid concentrations and lipoprotein composition. This study sought to investigate the resultant effect of obesity on cholesterol lipoprotein composition and circulating adiponectin concentrations in cats. Plasma glucose, lipids (triglyceride, cholesterol and free fatty acid), insulin and adiponectin concentrations, and cholesterol lipoprotein composition were measured and compared between body condition score (BCS) determined normal healthy control and obese cats. Although the obese group demonstrated higher levels of plasma cholesterol, glucose, and triglycerides, as compared to healthy controls, the difference was insignificant thus indicating that the BCS determined obese cats may have been overweight and not morbidly obese. Plasma insulin levels were significantly higher (25-30%) versus healthy control animals thereby possibly hinting at the ensuing emergence of obesity induced insulin resistance. However, the BCS determined obese cat demonstrated a significant reduction (p<0.05) in plasma adiponectin concentration and a significant increase (p<0.05) in LDL-cholesterol % as compared to age matched healthy control animals. This would indicate that changes in plasma adiponectin concentration and cholesterol lipoprotein composition may be good early indicators of obesity in cats.

Comparison of time-action profiles of insulin Glargine and NPH insulin in normal and diabetic dogs

Intermediate insulin injections are commonly used for glycemic control in insulin dependent diabetic dogs acting as a replacement for natural insulin. Neutral Protamin Hagedorn (NPH) insulin and insulin glargine are two types of injectable insulin preparations commonly used in humans. In our study, we investigated the time-action profiles of both aforementioned insulin preparations in normal dogs in order to determine whether co-administration of NPH and glargine would be of benefit to insulin dependent diabetic dogs as it is for humans suffering from insulin dependent diabetes. Time-action profiles of NPH insulin and insulin glargine in normal dogs demonstrated a clear difference between both insulin preparations confirming that NPH insulin is an intermediate-acting preparation whereas insulin glargine is a long-lasting preparation. In addition, co-administration of NPH insulin and insulin glargine resulted in tight glycemic control as compared to NPH insulin alone in insulin dependent diabetic dogs. However, co-administration result in hypoglycemia at the dosages tested.