Effects of selective and non-selective glucocorticoid receptor II antagonists on rapid-onset diabetes in young rats

Abstract

The blockade of glucocorticoid (GC) action through antagonism of the glucocorticoid receptor II (GRII) has been used to minimize the undesirable effects of chronically elevated GC levels. Mifepristone (RU486) is known to competitively block GRII action, but not exclusively, as it antagonizes the progesterone receptor. A number of new selective GRII antagonists have been developed, but limited testing has been completed in animal models of overt type 2 diabetes mellitus. Therefore, two selective GRII antagonists (C113176 and C108297) were tested to determine their effects in our model of GC-induced rapid-onset diabetes (ROD). Male Sprague-Dawley rats (∼ six weeks of age) were placed on a high-fat diet (60%), surgically implanted with pellets containing corticosterone (CORT) or wax (control) and divided into five treatment groups. Each group was treated with either a GRII antagonist or vehicle for 14 days after surgery: CORT pellets (400 mg/rat) + antagonists (80 mg/kg/day); CORT pellets + drug vehicle; and wax pellets (control) + drug vehicle. After 10 days of CORT treatment, body mass gain was increased with RU486 (by ∼20% from baseline) and maintained with C113176 administration, whereas rats given C108297 had similar body mass loss (∼15%) to ROD animals. Fasting glycemia was elevated in the ROD animals (>20 mM), normalized completely in animals treated with RU486 (6.2±0.1 mM, p<0.05) and improved in animals treated with C108297 and C113176 (14.0±1.6 and 8.8±1.6 mM, p<0.05 respectively). Glucose intolerance was normalized with RU486 treatment, whereas acute insulin response was improved with RU486 and C113176 treatment. Also, peripheral insulin resistance was attenuated with C113176 treatment along with improved levels of β-cell function while C108297 antagonism only provided modest improvements. In summary, C113176 is an effective agent that minimized some GC-induced detrimental metabolic effects and may provide an alternative to the effective, but non-selective, GRII antagonist RU486.

Figure 1. C113176 treatment maintains body mass while RU486 increases body mass with ROD treatment.

Animal body mass (g) were recorded every two days for 10 days as a measure of fold change from day 0, pellet surgery (A). Animal body mass on day 10 was measured as a percent change of body mass from day 0 (B). The dotted line (100%) represents no change in body mass from day 0. Arrow indicates that 2 days after pellet surgeries respective antagonists or vehicle were administered at 80 mg/kg/day to each treatment group. Bars that do not share similar letters denote statistical significance, p<0.05, one-way ANOVA using Tukey's post-hoc test. n = 7–10. All values are means ± SE.

Figure 2. Glucose intolerance and acute insulin response (AIR) is improved with RU486 and C113176 treatment.

Fasting (basal, 0 minutes) and stimulated blood glucose levels (mM) were measured at 5, 15, 30, 60, 90 and 120 minutes post oral glucose gavage (A). Glucose area under the curve (AUC) was calculated based on fasting blood glucose of individual animals (A′). Fasting (basal, 0 minutes) and glucose-stimulated insulin levels (ng/ml) were measured at 15, 30, 60, and 120 minutes post oral glucose gavage (B). Insulin area under the curve (AUC) was calculated based on fasting individual insulin levels within each group (B′). To measure insulin capacity acute insulin response (AIR) was measured by the difference in insulin levels between fasting insulin and 15 minutes post glucose gavage (C). Negative values represent a decrease in insulin response, indicating impairment in insulin secretion. Bars that do not share similar letters denote statistical significance, p<0.05, one-way ANOVA using Tukey's post-hoc. A student's unpaired t-test was performed between controls and ROD, C108297 and C113176 groups (C). n = 7–10. All values are means ± SE.

Figure 3. Peripheral insulin sensitivity and β-cell function is enhanced with RU486 and C113176 administration.

Percent change of blood glucose levels (mM) relative to individual fasting blood glucose at 5, 10, 20 and 30 minutes post insulin i.p. injection using a handheld glucometer (A). The glucose AUC was measured by the net inverse glucose AUC during the ITT (A′). HOMA-IR index was used to measure whole-body insulin resistance (B). HOMA-β index was used to measure pancreatic β-cell function (C). Bars that do not share similar letters denote statistical significance, p<0.05 one-way ANOVA using Tukey's post-hoc test. n = 7–10. All values are means ± SE.

Figure 4. Fat accumulation is normalized with RU486 in skeletal muscle and liver cross sections.

To determine fat content in skeletal muscle, tibialis anterior muscle was dissected and stained with a neutral lipid stain (Oil Red O) (A–E). Cross sections of liver were also stained with Oil Red O to measure lipid content (F–J).

Figure 5. 11β-HSD1 content in visceral fat is attenuated with RU486 treatment but not with C113176 or C108297 treatment.

No changes were found in lipolytic protein levels with treatment of antagonists. 11β-HSD1 content was measured in epididymal fat pads to represent visceral adipose tissue and expressed relative to α-tubulin content (A). CD36 protein levels were measured from epididymal fat pads (B) as well as adipose triglyceride lipase (ATGL) (C) and hormone-sensitive lipase (HSL) (D) protein levels as markers of lipolytic adipose tissue activity and expressed relative to loading control. Bars that do not share similar letters denote statistical significance, p<0.05 one-way ANOVA using Tukey's post-hoc test. n = 5–6. All values are means ± SE.

Figure 6. Glucose stimulated insulin secretion (GSIS) was normalized with RU486 and C113176 treatment.

GSIS in isolated islets was measured in low (2.8 mM) and high (16.7 mM) glucose media for 1-hour incubations expressed as ng/ml/islet/hour. Bars that do not share similar letters denote statistical significance, p<0.05 one-way ANOVA using student's unpaired t-test. n = 3–7. All values are means ± SE.