Daigo Azakami

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.

Activities of enzymes in the malate-aspartate shuttle in the peripheral leukocytes of dogs and cats.

The activities of the enzymes involved in the malate–aspartate shuttle and the expression of malate dehydrogenase (MDH), a rate-limiting enzyme in the NADH shuttle that produces ATP in glucose metabolism in leukocytes, were determined to investigate the differences in this shuttle system in the peripheral leukocytes of dogs and cats. There were no significant differences between dogs and cats in plasma glucose, immunoreactive insulin, free fatty acid or triglyceride concentrations. The activities of cytosolic and mitochondrial MDH and of mitochondrial glutamate dehydrogenase (GLDH) in canine leukocytes were significantly higher than in feline leukocytes. High activities of MDH in canine leukocytes were confirmed by RT-PCR analysis on the total RNA extracted from leukocytes. It was concluded that there were significant differences between dogs and cats in the NADH shuttle system.

Identification of tumor-initiating cells in a canine hepatocellular carcinoma cell line

Tumor-initiating cells (TICs) or cancer stem cells (CSCs), a small subset of tumor cells, are involved in tumor initiation, progression, recurrence and metastasis. In human hepatocellular carcinoma (HCC), TICs are enriched with cell surface markers and have the ability to self-renew and differentiate tumors at a high frequency. We established a canine HCC cell line, HCC930599, and analyzed it for stem and progenitor cell marker expression using flow cytometry. HCC930599 showed high CD44 and CD29, moderate CD90, and low CD133, CD34, CD24, CD117, and CD13 expression. CD90(+)CD44(+) and CD90(-)CD44(+) cells were characterized using the in vitro sphere assay and an in vivo transplant model. CD90(+)CD44(+) cells acquired enhanced self-renewal capacity, proliferative activity and tumourigenicity compared with CD90(-)CD44(+) cells, suggesting that TICs exist in the HCC930599 cell line and that CD90 is a marker for enriched TICs. Understanding TIC characteristics may help elucidate hepatic carcinogenesis and HCC therapy development.

Canine epidermal langerhans cells express α and γ but not β chains of high-affinity IgE receptor

Epidermal Langerhans cells (LC) express a high-affinity receptor for IgE (FcεRI), consisting of two chains (α and γ chains) in humans that allows LC to perform Fc receptor-mediated uptake of allergens. We found that canine LC express α and γ chains but not β chain of FcεRI, identical to human but not to mouse LC, which do not express functional FcεRI (only γ chain is expressed). This finding indicates that canine LC have FcεRI-mediated function similar to or identical to human LC, raising the possibility that canine species provides a better model than mouse to understand the pathogenesis of human atopic dermatitis and investigate the therapeutic effect of drugs.

Genetic variants of the unsaturated fatty acid receptor GPR120 relating to obesity in dogs

G protein-coupled receptor (GPR) 120 is an unsaturated fatty acid receptor, which is associated with various physiological functions. It is reported that the genetic variant of GPR120, p.Arg270His, is detected more in obese people, and this genetic variation functionally relates to obesity in humans. Obesity is a common nutritional disorder also in dogs, but the genetic factors have not ever been identified in dogs. In this study, we investigated the molecular structure of canine GPR120 and searched for candidate genetic variants which may relate to obesity in dogs. Canine GPR120 was highly homologous to those of other species, and seven transmembrane domains and two N-glycosylation sites were conserved. GPR120 mRNA was expressed in lung, jejunum, ileum, colon, hypothalamus, hippocampus, spinal cord, bone marrow, dermis and white adipose tissues in dogs, as those in mice and humans. Genetic variants of GPR120 were explored in client-owned 141 dogs, resulting in that 5 synonymous and 4 non-synonymous variants were found. The variant c.595C>A (p.Pro199Thr) was found in 40 dogs, and the gene frequency was significantly higher in dogs with higher body condition scores, i.e. 0.320 in BCS4–5 dogs, 0.175 in BCS3 dogs and 0.000 in BCS2 dogs. We conclude that c.595C>A (p.Pro199Thr) is a candidate variant relating to obesity, which may be helpful for nutritional management of dogs.

Analyses of a satiety factor NUCB2/nesfatin-1; gene expressions and modulation by different dietary components in dogs

Nesfatin-1 is an anorexic peptide derived from a precursor, nucleobindin-2 (NUCB2), which is distributed in various organs, coexists with ghrelin in the gastric X/A-like cells and closely relates to an appetite control in rodents and humans. Nesfatin-1 may be a significant factor addressing the satiety also in veterinary medicine, however, there are few reports about nesfatin-1 in dogs. In the present study, we detected canine NUCB2/nesfatin-1 mRNA in various tissues, especially abundant in pancreas, gastrointestinal tracts, testis and cerebellum. We examined circulating nesfatin-1 concentrations and NUCB2/nesfatin-1 mRNA expressions in upper gastrointestinal tracts (gastric corpus, pyloric antrum and duodenum) in dogs fed on different types of diets. Plasma nesfatin-1 concentrations in the dogs were approximately 4 ng/ml and they did not change after feeding through the study, however, NUCB2/nesfatin-1 mRNA expressions in pyloric antrum were 1.84-fold higher in the dogs fed on a High fiber/High protein diet (P<0.001), 1.48-fold higher in the dogs fed on a High fat/Low protein diet (P<0.05) and 1.02-fold higher in the dogs fed on a Low fat/High carbohydrate diet (not significant) comparing to those on a control diet. It was concluded that High fiber/High protein and High fat/Low protein diets increased NUCB2/nesfatin-1 production in canine gastrointestinal tracts. These results may set the stage for further investigations of canine NUCB2/nesfatin-1, which may relate to satiety effects in dogs.

Molecular Characterization of the Cytidine Monophosphate-N-Acetylneuraminic Acid Hydroxylase (CMAH) Gene Associated with the Feline AB Blood Group System

Cat’s AB blood group system (blood types A, B, and AB) is of major importance in feline transfusion medicine. Type A and type B antigens are Neu5Gc and Neu5Ac, respectively, and the enzyme CMAH participating in the synthesis of Neu5Gc from Neu5Ac is associated with this cat blood group system. Rare type AB erythrocytes express both Neu5Gc and Neu5Ac. Cat serum contains naturally occurring antibodies against antigens occurring in the other blood types. To understand the molecular genetic basis of this blood group system, we investigated the distribution of AB blood group antigens, CMAH gene structure, mutation, diplotypes, and haplotypes of the cat CMAH genes. Blood-typing revealed that 734 of the cats analyzed type A (95.1%), 38 cats were type B (4.9%), and none were type AB. A family of three Ragdoll cats including two type AB cats and one type A was also used in this study. CMAH sequence analyses showed that the CMAH protein was generated from two mRNA isoforms differing in exon 1. Analyses of the nucleotide sequences of the 16 exons including the coding region of CMAH examined in the 34 type B cats and in the family of type AB cats carried the CMAH variants, and revealed multiple novel diplotypes comprising several polymorphisms. Haplotype inference, which was focused on non-synonymous SNPs revealed that eight haplotypes carried one to four mutations in CMAH, and all cats with type B (n = 34) and AB (n = 2) blood carried two alleles derived from the mutated CMAH gene. These results suggested that double haploids selected from multiple recessive alleles in the cat CMAH loci were highly associated with the expression of the Neu5Ac on erythrocyte membrane in types B and AB of the feline AB blood group system.

Tumor suppressor REIC/DKK-3 and co-chaperone SGTA: Their interaction and roles in the androgen sensitivity

REIC/DKK-3 is a tumor suppressor, however, its intracellular physiological functions and interacting molecules have not been fully clarified. Using yeast two-hybrid screening, we found that small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), known as a negative modulator of cytoplasmic androgen receptor (AR) signaling, is a novel interacting partner of REIC/DKK-3. Mammalian two-hybrid and pull-down assay results indicated that the SGTA-REIC/DKK-3 interaction involved the N-terminal regions of both REIC/DKK-3 and SGTA and that REIC/DKK-3 interfered with the dimerization of SGTA, which is a component of the AR complex and a suppressor of dynein motor-dependent AR transport and signaling. A reporter assay in human prostate cancer cells that displayed suppressed AR signaling by SGTA showed recovery of AR signaling by REIC/DKK-3 expression. Considering these results and our previous data that REIC/DKK-3 interacts with the dynein light chain TCTEX-1, we propose that the REIC/DKK-3 protein interferes with SGTA dimerization, promotes dynein-dependent AR transport and then upregulates AR signaling.

Molecular cloning and tumour suppressor function analysis of canine REIC/Dkk-3 in mammary gland tumours

REIC/Dkk-3, a member of the human Dickkopf (Dkk) family, plays a role as a suppressor of growth in several human cancers. In this study, the tumour suppression function of canine REIC/Dkk-3 was investigated. The full-length open reading frame of the canine REIC/Dkk-3 homologue was cloned and the tissue distribution of REIC/Dkk-3 mRNA was determined, along with the subcellular localisation of the REIC/Dkk-3 protein in canine cancer cell lines. Expression of REIC/Dkk-3 was lower in mammary gland tumours and in canine mammary carcinoma cell lines than in normal mammary gland tissue. Overexpression of REIC/Dkk-3 induced apoptosis in canine mammary carcinoma cell lines. These results show that expression of REIC/Dkk-3 is downregulated in canine mammary tumours and that one of the functions of this gene is induction of apoptosis.

Transepidermal water loss in cats: comparison of three differently clipped sites to assess the influence of hair coat on transepidermal water loss values

BACKGROUND The measurement of transepidermal water loss (TEWL) is one of the parameters that can be used to assess skin barrier function. The variability and reliability of TEWL measurements in dogs have been controversial, and the hair coat has been considered as one of the factors that may cause variation of TEWL values. HYPOTHESIS/OBJECTIVES The aims of the study were to establish a suitable procedure for measuring feline TEWL, to evaluate the influence of hair coat on TEWL measurements and to assess variations of TEWL at different anatomical sites. METHODS Transepidermal water loss was measured using a closed-chamber evaporimeter, the VapoMeter(®). We compared three adjacent sites in the groin area of 10 clinically normal, domestic short hair cats. One site was unclipped, the second was trimmed with scissors and the third was shaved using electric clippers. Values of TEWL were obtained for 48 h after trimming with scissors and clippers. Five sites were clipped (upper back, lumbar back, lateral thigh, axillae and groin), and the TEWL was measured. RESULTS The mean and SD of TEWL values of the clipper-trimmed site were the smallest, followed in order by the site trimmed with scissors and the unclipped site. The TEWL values were statistically constant in the clipper-trimmed site, while the values in the unclipped sites were not. There was no statistically significant difference in TEWL values between all of the anatomical sites except for the axillae. CONCLUSIONS AND CLINICAL IMPORTANCE Hair clipping of sites with electric clippers is recommended for TEWL measurement in cats.