Pyometra-associated insulin resistance assessment by insulin binding assay and tyrosine kinase activity evaluation in canine muscle tissue

Abstract

Diestrus is associated with insulin resistance in bitches and pyometra can further impair insulin sensitivity. This study aimed to compare insulin sensitivity, insulin binding, and tyrosine kinase activity in bitches in anestrus, diestrus, or with pyometra. Patients submitted to elective ovariohysterectomy were divided into anestrus (n = 11) or diestrus (n = 13) according to reproductive history, vaginal cytology, and uterine histology. The group pyometra (n = 8) included bitches diagnosed with the disease based on clinical presentation and abdominal ultrasound findings and further confirmed by uterine histopathology. All patients were submitted to an intravenous glucose tolerance test (IVGTT) before ovariohysterectomy, and rectus abdominis muscle samples were collected during surgery for plasmatic membrane suspension preparation. Muscle-membranes were submitted to cold saturation insulin binding assay for dissociation constant (Kd) and maximum binding capacity (Bmax) determination, as well as exogenous substrate Poly (Glu: Tyr 4:1) phosphorylation assay for basal tyrosine kinase evaluation. Bitches with pyometra showed higher basal insulin (P < 0.001) and higher area under the curve (AUC) for insulin (P = 0.01) and glucose (P < 0.001) response during the IVGTT in comparison with bitches in anestrus or diestrus. Diestrus (P < 0.0001) and pyometra (P = 0.001) were associated with reduced tyrosine kinase activity in comparison with anestrus. No differences were documented in Kd and Bmax results for the low-affinity/high-capacity insulin receptors; however, high-affinity/low-capacity insulin receptors showed higher Kd and Bmax results in bitches in diestrus or with pyometra (P < 0.05) in comparison with anestrus. Despite the pyometra group showed the highest Kd values (P < 0.01), its Bmax results did not differ from the diestrus group (P > 0.05). Diestrus' higher Kd values and reduced tyrosine kinase activity in muscle tissue were compensated by increased total insulin binding capacity. Absent differences in IVGTT results between diestrus and anestrus bitches corroborate this finding. However, in bitches with pyometra, the highest Kd values were not compensated by increased total insulin binding capacity. This finding was associated with insulin resistance and glucose intolerance in IVGTT results. Moreover, pyometra resolution restored insulin sensitivity and glucose tolerance. These features can play a key role in pyometra-associated CDM, as well as in diabetic remission after pyometra resolution.

Keywords: Estrous cycle; Glucose tolerance; Insulin receptor; Insulin resistance; Tyrosine kinase receptors.