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

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.

Redox and metabolic strategies developed by anterior and posterior gills of the crab Neohelice granulata after short periods of hypo- or hyper-osmotic stress.

The aim of this study was to identify the response pattern of redox balance, Na+/K+ATPase activity and HSP70 expression in the posterior and anterior gills of the crab Neohelice granulata submitted to hypo- or hyper-osmotic stress for 1 h and 6 h. After 1 h of either type of osmotic stress, there was an increase in catalase activity, but a decrease in GSSG/GSH ratio (oxidized to reduced glutathione ratio) and Na+/K+ATPase activity in both gill sets. H2O2 levels decreased only in the posterior gills. H2O2 levels and Na+/K+ATPase activity remained reduced after 6 h of exposure to either type of osmotic stress in both gill sets. The GSSG/GSH ratio returned to initial levels after 6 h of hyper-osmotic stress, whereas it increased 10 times in both gill sets after hypo-osmotic stress. Furthermore, HSP70 protein expression increased in posterior gills after 6 h of hypo-osmotic stress. H2O2 levels in tank water decreased after hypo-osmotic challenge and increased after 6 h of hyper-osmotic stress, indicating increased H2O2 excretion. Therefore, N. granulata gills have redox, metabolic and molecular strategies to deal with rapid osmotic challenges, an important environmental parameter that influences juvenile and adult crab distribution and abundance within different populations.

Glyceroneogenesis in the hepatopancreas of the crab Neohelice granulata: Diet, starvation and season effects.

We determined the activity of glyceroneogenesis from [2-14C]-pyruvate, the phosphoenolpyruvate carboxykinase activity, [2-14C]-pyruvate oxidation and total lipid levels in the hepatopancreas of the crab Neohelice granulata fed with a carbohydrate-rich (HC) diet or a high-protein (HP) diet and then subjected to 5weeks of starvation, in summer and winter, to determine whether the seasonal adjustments of lipid metabolism to food scarcity are modulated by the composition of the diet previously given to the crabs. The results demonstrated that glyceroneogenesis is an active pathway in N. granulata hepatopancreas, and is regulated by seasonal variations, diet composition and starvation. This study showed that in summer the increase in the hepatopancreas glyceroneogenesis activity is among the strategies used by N. granulata fed an HP diet, to maintain the triglyceride/fatty acid cycle during starvation, a normal condition in the biological cycle of this crab. However, the administration of an HC diet reduced the glyceroneogenesis capacity in response to starvation in summer. In winter, the decrease in the glyceroneogenesis capacity in both fed (HP and HC diets) and starved crabs seems to be a strategy to reduce energy consumption and/or requirement. In contrast to the summer results, the incorporation of [2-14C]-pyruvate into 14CO2 was markedly higher in both diet (HC and HP) groups and in starved crabs during the winter. Four decades after the first study describing the glyceroneogenesis pathway in rat white adipose tissue, this pathway is evidenced for the first time in a crustacean.