Eri Onozawa

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.

The canine prostate cancer cell line CHP‐1 shows over‐expression of the co‐chaperone small glutamine‐rich tetratricopeptide repeat‐containing protein α

Although androgen therapy resistance and poor clinical outcomes are seen in most canine prostate cancer cases, there are only a few tools for analysing canine prostate cancer by using a cell biological approach. Therefore, to evaluate androgen-independent neoplastic cell growth, a new canine prostate cancer cell line (CHP-1) was established in this study. CHP-1 over-expressed the co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), which is over-expressed in human androgen-independent prostate cancer. The CHP-1 xenograft also showed SGTA over-expression. Although CHP-1 shows poor androgen receptor (AR) signalling upon dihydrotestosterone stimulation, forced expression of AR enabled evaluation of AR signalling. Taken together, these results suggest that CHP-1 will be a useful model for investigating the pathogenesis of androgen-dependent and androgen-independent canine prostate cancer.

Cholesterol concentrations in lipoprotein fractions separated by anion-exchange–high-performance liquid chromatography in healthy dogs and dogs with hypercholesterolemia

Anion-exchange (AEX)-high-performance liquid chromatography (HPLC) for measurement of cholesterol can be used to separate serum lipoproteins (high-density lipoprotein (HDL); low-density lipoprotein (LDL); intermediate-density lipoprotein (IDL); very-low-density lipoprotein (VLDL)) in humans. However, AEX-HPLC has not been applied in veterinary practice. We had three objectives: (i) the validation of AEX-HPLC methods including the correlation of serum cholesterol concentration in lipoprotein fraction measured by AEX-HPLC and gel permeation-HPLC (GP-HPLC) in healthy dogs and those with hypercholesterolemia was investigated; (ii) the reference intervals of lipoprotein fractions measured by AEX-HPLC from healthy dogs (n=40) was established; (iii) lipoprotein fractions from the serum of healthy dogs (n=12) and dogs with hypercholesterolemia (n=23) were compared. Analytic reproducibility and precision of AEX-HPLC were acceptable. Positive correlation between serum concentrations of total cholesterol (Total-Chol), HDL cholesterol (HDL-Chol), LDL cholesterol (LDL-Chol)+IDL cholesterol (IDL-Chol), and VLDL cholesterol (VLDL-Chol) was noted for AEX-HPLC and GP-HPLC in healthy dogs and dogs with hypercholesterolemia. Reference intervals measured by AEX-HPLC for serum concentrations of Total-Chol, HDL-Chol, and LDL-Chol were determined to be 2.97-9.32, 2.79-6.57, 0.16-3.28mmol/L (2.5-97.5% interval), respectively. Furthermore, there was significant difference in lipoprotein profiles between healthy and dogs with hypercholesterolemia. These results suggest that AEX-HPLC can be used to evaluate lipoprotein profiles in dogs and could be a new useful indicator of hyperlipidemia in dogs.

Identification of tumor-initiating cells derived from two canine rhabdomyosarcoma cell lines

Cancer stem cells or tumor-initiating cells (TICs) are a small subpopulation of cells that have the capacity to self-renew, differentiate and initiate tumors. These cells may function in tumor initiation, aggression and recurrence. Whether spheres derived from canine rhabdomyosarcoma cells have stem cell-like properties is unclear. We induced sphere formation in the canine rhabdomyosarcoma cell lines, CMS-C and CMS-J, and characterized the spheres in vitro and in vivo. Sphere-forming cells were more resistant to vincristine, mitoxantrone and doxorubicin than adherent cells. Xenograft transplantation demonstrated that 1 × 103 sphere-forming cells derived from CMS-C were sufficient for tumor formation. The sphere assay showed that the sphere-forming cells were present in these tumors. These results suggest that the spheres derived from canine rhabdomyosarcoma cells may possess characteristics of TICs. This study provides the foundation for elucidating the contribution of TICs to rhabdomyosarcoma tumorigenesis.

Primary T-cell high-grade lymphoma of the feline uterus

A 12-year-old female American shorthair cat presented with a one-month history of hematuria and general lethargy. Abdominal ultrasonography revealed complete thickening of the left uterine wall. At a diagnostic laparotomy, a large mass arising from the left uterine horn was discovered, and ovariohysterectomy was performed. Histological diagnosis revealed a T-cell high-grade lymphoma of the uterus. After the ovariohysterectomy, the patient achieved complete remission and was maintained by combination chemotherapy from 14 days after surgery. However, relapse occurred in the urinary bladder wall on day 287, and the patient died of postrenal acute renal failure on day 310. This is the first report of a feline case of primary uterine lymphoma that was treated with ovariohysterectomy followed by systemic chemotherapy.

R132 mutations in canine isocitrate dehydrogenase 1 (IDH1) lead to functional changes

Glioma is the second most common intracranial neoplasia in dogs, but the pathogenic mechanisms remain unclear. In humans, isocitrate dehydrogenase 1 (IDH1) is frequently mutated in gliomas. Although almost all human IDH1 mutations have been identified as involving the Arg132 codon, few studies have reported structural, functional, and mutational information for canine IDH1. Therefore, in this study, we cloned the canine IDH1 homologue and used PCR mutagenesis to substitute the wildtype (WT) Arg132 with His (R132H) or Ser (R132S). WT and mutated IDH1 were overexpressed in HeLa cells, and their presence was confirmed by immunoblotting and immunocytochemistry using mutation-specific antibodies. The IDH1 activity between WT, R132H, and R132S transfectants was compared by measuring the production of NADH and NADPH. NADPH production in R132H and R132S transfectants was lower than that in WT, but NADH levels were not significantly different. Finally, we detected increased expression of hypoxia inducible factor 1 alpha (HIF-1α) in the R132H and R132S transfectants. These results indicated that the canine IDH1 Arg132 mutation has the potential to induce carcinogenesis in canine somatic cells.

Time-action profiles of insulin degludec in healthy dogs and its effects on glycemic control in diabetic dogs

Insulin degludec (IDeg) is a new insulin formulation that facilitates long-term control of glucose level in humans. In this study, we investigated the effects of IDeg on glycemic control in dogs. Its time-action profiles were monitored in healthy dogs using an artificial pancreas apparatus under euglycemic conditions. At 9.0–13.5 hr post-IDeg injection, an indistinct peak of glucose level was detected. Moreover, the action of IDeg was persistent for >20 hr. Both IDeg and neutral protamine Hagedorn insulin (NPH) lowered blood glucose concentrations in diabetic dogs, but IDeg caused postprandial hyperglycemia and a somewhat lower preprandial glucose level than that caused by NPH. IDeg might be ineffective in concurrently preventing postprandial hyperglycemia and preprandial hypoglycemia in a single-agent administration.

Properties of the feline tumour suppressor reduced expression in immortalized cells (REIC/Dkk‐3)

Reduced expression in immortalized cells (REIC/Dkk-3), a member of the human Dickkopf (Dkk) family, is a growth suppressor in human and canine mammary tumours. Mammary gland tumours are common neoplasms with high malignancy in female cats. The purpose of this study was to clone the feline REIC/Dkk-3 homolog, investigate its expression in cell lines established from feline mammary gland tumours, and test its tumour suppressor function. Western blot analysis revealed that expression of the REIC/Dkk-3 protein was reduced in feline mammary carcinoma cell lines. Forced expression of REIC/Dkk-3 induced apoptosis in feline mammary tumour cell lines. These results demonstrate that REIC/Dkk-3 expression, which is downregulated in feline mammary tumour cell lines, results in the induction of apoptosis in these cells. Our findings suggest that feline REIC/Dkk-3 represents a potential molecular target for the development of therapies against feline mammary cancers.

Evaluation of newly developed veterinary portable blood glucose meter with hematocrit correction in dogs and cats

This study evaluated the accuracy of a newly developed veterinary portable blood glucose meter (PBGM) with hematocrit correction in dogs and cats. Sixty-one dogs and 31 cats were used for the current study. Blood samples were obtained from each dog and cat one to six times. Acceptable results were obtained in error grid analysis between PBGM and reference method values (glucose oxidation methods) in both dogs and cats. Bland–Altman plot analysis revealed a mean difference between the PBGM value and reference method value of −1.975 mg/dl (bias) in dogs and 1.339 mg/dl (bias) in cats. Hematocrit values did not affect the results of the veterinary PBGM. Therefore, this veterinary PBGM is clinically useful in dogs and cats.

The effect of 1 year of trilostane treatment on peripheral lymphocyte subsets in dogs with pituitary-dependent hyperadrenocorticism

This study investigated the changes in lymphocyte subsets during the trilostane medication of Pituitary-dependent hyperadrenocorticism (PDH) dogs. The cortisol level and lymphocyte subsets of eight dogs with PDH were monitored 0, 1, 3, 6, 9 and 12 months after the initiation of trilostane treatment. White blood cells (WBC), lymphocytes, CD3+ (T lymphocyte), CD4+ (helper T lymphocyte), CD8+ (cytotoxic T lymphocyte) and CD21+ (B lymphocyte) cells were measured. Although the post-ACTH stimulation test cortisol level was significantly lower during trilostane treatment, changes in the CD3+, CD4+, CD8+ and CD21+ counts were not observed. Meanwhile, significant decrease was observed in WBC counts during trilostane treatment. These indicate that long-term trilostane treatment has little effect on the lymphocyte subsets in PDH dogs.