Toshinori Sako

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.

A comparison of the activities of certain enzymes related to energy metabolism in leukocytes in dogs and cats.

The activities of Na>+,K>+-ATPase in plasma membrane, of cytosolic enzymes and of glutamate dehydrogenase (GlGD) in mitochondria were measured in leukocytes (WBC) from dogs and cats to clarify the differences in energy metabolism in these cells. Feline WBC had significantly higher activities of hexokinase (HK), pyruvate kinase (PK) and LDH with pyruvate as substrate than did canine WBC. Canine WBC had significantly higher activities of glucokinase (GK) and GlDH than did feline WBC. Feline WBC had unique characteristics of energy metabolism in that the activities of the cytosolic enzymes under anaerobic conditions were significantly higher than those in canine WBC. It therefore appears that there are distinct differences in glucose metabolism in WBC between dogs and cats. WBC enzyme activities are considered to reflect the metabolic state in the whole body of the animal. It is therefore suggested that changes in the activities of certain glycolytic enzymes in WBC may be useful as a diagnostic indicator in some types of metabolic disease in dogs and cats.

D-glucose transport and glycolytic enzyme activities in erythrocytes of dogs, pigs, cats, horses, cattle and sheep.

The activities of D-glucose transport (D-GT) and the glycolytic enzymes hexokinase (HK) and pyruvate kinase (PK), were measured in the erythrocytes of dogs, pigs, cats, horses, cattle and sheep. The erythrocytes of dogs had the highest activities of D-GT, HK and PK, significantly higher than the activities in the erythrocytes of the herbivores. The activities of D-GT and HK in cat erythrocytes were significantly lower than in those of dogs. The differences between the activities of D-GT in the erythrocytes of the different species followed the differences in activities of HK but not those in the activities of PK or in the blood glucose concentrations. It is considered that the activity of HK provides a convenient measurement of the relative rates of glucose oxidation in erythrocytes.

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.

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.

Comparison of insulin signaling gene expression in insulin sensitive tissues between cats and dogs.

Diabetes mellitus (DM) is a common endocrine disease in cats and dogs with increasing prevalence. Type 1 DM appears to be the most common form of diabetes in dogs whereas Type 2 DM prevails for cats. Since insulin resistance is more frequently encountered in cats than dogs, our laboratory was interested in determining whether differences at the insulin signaling pathway level and differences in glucose and lipid metabolism could be observed between cats and dogs. Insulin resistance has been positively correlated to insulin signaling pathway abnormalities. As such, this study measured insulin receptor substrate-1 (IRS-1), insulin receptor substrate-2 (IRS-2), and phosphatidylinositol 3-kinase (PI3-K) P-85α mRNA expression levels in classical insulin-responsive sensitive tissues (liver, skeletal muscle, and abdominal fat) and peripheral leukocytes between cats and dogs by qRT-PCR. Different tissues were sampled because it is currently unknown where insulin-resistance arises from. In addition, enzymes involved in glucose and lipid metabolism, malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH) and fatty acid synthase (FAS) were also assessed since glucose and lipid metabolism differs between cats and dogs. Overall, IRS-1, IRS-2, PI3-K, MDH, G6DPH, and FAS mRNA tissue expression profiles demonstrated different levels of expression, in various tissues for both canines and felines, which was expected. No distinct expression pattern emerged; however, differences were noted between canines and felines. In addition, IRS-1, IRS-2, PI3-K, MDH, G6DPH, and FAS mRNA expression was significantly higher in canine versus feline tissues, including peripheral leukocytes. Remarkable differences in insulin signaling gene expression between felines and canines indicate that cats may have an underlying low insulin sensitivity level due to low IRS-1, IRS-2, and PI3-K P-85α mRNA expression levels which would predispose cats to develop insulin resistance. Moreover, differences in glucose and lipid metabolism related gene expression (MDH, G6DPH, and FAS) demonstrate that felines have an overall lower metabolic rate in various tissues which may be attributed to overall lower insulin signaling gene expression and a lack of physical activity as compared to canines. Therefore, a combination of genetic and environmental factors appears to make felines more prone to suffer from insulin resistance and type 2 DM than canines.

Comparison of glucokinase activities in the peripheral leukocytes between dogs and cats.

Activities of hexokinase (HK), glucokinase (GK) and pyruvate kinase (PK), were measured. The expression of GK mRNA was investigated using reverse transcription-polymerase chain reaction (RT-PCR) in leukocytes (WBC) of dogs and cats. No significant differences between dogs and cats were found in concentrations of blood glucose and plasma insulin. Dog WBC showed GK activities and the specific fragment with predicted size of 574 bp containing conserved region including glucose- and ATP-binding domains of GK as determined with RT-PCR. However, in cat WBC, the activities and specific fragment of GK were absent. After fasting, the activities and gene expression of GK decreased greatly in the dog WBC. The cat WBC had significantly higher activities of HK and PK than dog WBC.

Time-action profiles of insulin detemir in normal and diabetic dogs.

Insulin detemir is the first member of a new class of long-acting soluble insulin analogues capable of maintaining the basal level of insulin in humans. In this preliminary study, we investigated the time-action profiles of insulin detemir in normal and diabetic dogs since the use of insulin detemir in canines has yet to be determined. Eight animals were used in our study (three normal and five insulin dependent diabetic dogs). Time-action profiles of insulin detemir were monitored in normal dogs using an artificial pancreas apparatus under euglycemic condition. Blood sampling was performed at 2h intervals post feeding, with insulin administration, in insulin dependent diabetic dogs. Time-action profiles of insulin detemir, in normal dogs, demonstrated that insulin detemir is a long-lasting preparation similar to what has been observed in humans. A pronounced peak was detected at 8-10h while the glucose-lowering effect lasted for over 24h after insulin injection, thus illustrating its longer prolonged peak activity time. Furthermore, intensive glycemic control was achieved with insulin detemir in insulin dependent diabetic dogs, using a lower dosage than NPH insulin and insulin glargine therapeutic doses. Our results indicate that insulin detemir has a greater effect than either NPH insulin or insulin glargine in canines, requiring a lower dose than either insulin preparation. However, using insulin detemir also carries a higher risk of inducing hypoglycemia as compared to either NPH insulin or insulin glargine.

Diagnostic significance of serum glycated albumin in diabetic dogs

Measurements of serum fructosamine, glycated hemoglobin, and glycated albumin (GA) are increasingly used to complement serum glucose concentration for better management of diabetes mellitus. Fructosamine tests are currently not performed in veterinary medicine in Japan. As such, the measurement of GA may serve as a replacement test. Therefore, in the current study, serum GA and fructosamine were evaluated for a positive correlation in dogs, and, depending on the correlation, a reference range of GA percentage would also be determined from healthy control dogs. The degree of glycemic control in diabetic dogs was determined by fructosamine concentration. A positive correlation between GA and fructosamine was observed with both normal and diabetic animals. In addition, the reference interval of serum GA percentage in control dogs was determined to be 11.4–11.9% (95% confidence interval). Interestingly, no significant difference in serum GA percentages was observed between samples from diabetic dogs with excellent glycemic control and control dogs. However, good, fair, and poor glycemic control diabetic dogs resulted in a significant increase in serum GA percentages in comparison with control dogs. These results suggest that serum GA may be a useful diagnostic indicator, substituting for fructosamine, to monitor glycemic control in diabetic dogs.

Comparison of Activities of Enzymes Related to Energy Metabolism in Peripheral Leukocytes and Livers between Holstein Dairy Cows and ICR Mice

Activities of enzymes related to energy metabolism and isoenzyme patterns of lactate dehydrogenase (LDH) were determined in peripheral leukocytes and livers of Holstein dairy cows and Institute of Cancer Research (ICR) mice. In dairy cow liver, activities of enzymes in glycolysis, malate–aspartate shuttle and lipogenesis were lower, but activities of glucose-6-phosphatase in gluconeogenesis were higher than those in mouse liver. Glucokinase activities were below detection limit in leukocytes and liver of the cows. Dairy cow leukocytes and liver showed the isoenzyme patterns with dominance of LDH-1, -2 and-3, whereas mouse leukocytes and liver showedthat LDH-5 was dominant. The LDH isoenzyme patterns were very similar between leukocytes and liver in each animal species. Some enzymes in leukocytes may reflect those enzymes activities in liver and be a useful indicator for energy metabolism in animals.