Animals and surgery
8 week old female C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine) were housed with a 12 h light-dark cycle. The mice were subjected to either a bilateral OVX or a sham operation under anesthesia with 1.2% avertin solution (i.p.). After a recovery period of 2 weeks, mice were divided into 6 treatment groups as follows: 1) sham+vehicle, 2) sham+STZ+vehicle, 3) OVX+STZ+vehicle, 4) OVX+STZ+CE, 5) OVX+STZ+BZA, 6) OVX+STZ+CE+BZA. Diabetes was induced with 50 mg/kg STZ (dissolved in 50 mM citrate buffer, pH 4.5) or with citrate buffer alone injected intraperitoneally for 5 consecutive days. All compounds were administered orally by gavage to mice once daily for 5 weeks with vehicle (saline, 2% Tween 80, 0.5% methylcellulose), CE 2.5 mg/kg, BZA 3 mg/kg or CE 2.5 mg/kg +BZA 3 mg/kg in the vehicle solution. The administered dosages of CE and BZA were chosen to ensure optimal maintenance of the estrogen action in the absence of uterine growth. All mice received phytoestrogen-free HFD (TD04059, 52% Kcal from anhydrous milk fat, Harlan Teklad, Madison, WI, USA) and water ad libitum during the experimental period. At the end of the study, mice were euthanized by an overdose of avertin, and blood was collected by cardiac puncture. All animal work was performed in compliance with the Institutional Animal Care and Use Committee of Northwestern University and in accordance to NIH guidelines.
Glucose and insulin measurements
Random-fed blood glucose was measured from blood obtained from the tail vein using a OneTouch Ultra 2 glucose meter (LifeScan, Inc., Milpitas, CA). Hyperglycemia was defined as a non-fasting blood glucose level ≥200 mg/dL. The cumulative incidence of diabetes was calculated as a percentage of hyperglycemic mice at each time point. Oral glucose tolerance test (OGTT) was performed at 3 weeks after STZ treatment. Mice were fasted overnight (16 h), and a glucose load (2 g/kg) was administered orally. Blood glucose and plasma insulin levels were measured from the tail vein at 15, 30, 60 and 90 min after administration of glucose. The area under the curve (AUC) for glucose and insulin was calculated for each group of animals during OGTT. Following euthanasia, plasma was separated by centrifugation at 3000 g for 20 min at 4°C and used for the determination of insulin levels using an ELISA kit (Millipore). The pancreas was isolated, homogenized in acidified ethanol, extracted overnight at 4°C, and centrifuged. The insulin content of the supernatant was determined using an ELISA kit (Millipore) and expressed in ng/mg pancreas.
Data were analyzed by one-way ANOVA using SAS software for Windows release 9.2 (SAS Institute Inc., Cary, NC, USA) on the W32_VSHOME platform. To test for differences in uterine weights among the treatment groups, analysis of covariance (ANCOVA) with final body mass as a covariate was used. Homogeneity of regression assumptions of the ANCOVA model were tested and met in each analysis. Differences in cumulative incidence of diabetes were determined by the log-rank test. The Least Squares Means option using a Tukey–Kramer adjustment was used for multiple comparisons among the treatment groups. Data were presented as the mean ± SEM. P values <0.05 were considered statistically significant.