One week of continuous corticosterone exposure impairs hepatic metabolic flexibility, promotes islet β-cell proliferation, and reduces physical activity in male C57BL/6 J mice

Abstract

Clinical glucocorticoid use, and diseases that produce elevated circulating glucocorticoids, promote drastic changes in body composition and reduction in whole body insulin sensitivity. Because steroid-induced diabetes is the most common form of drug-induced hyperglycemia, we investigated mechanisms underlying the recognized phenotypes associated with glucocorticoid excess. Male C57BL/6 J mice were exposed to either 100ug/mL corticosterone (cort) or vehicle in their drinking water. Body composition measurements revealed an increase in fat mass with drastically reduced lean mass during the first week (i.e., seven days) of cort exposure. Relative to the vehicle control group, mice receiving cort had a significant reduction in insulin sensitivity (measured by insulin tolerance test) five days after drug intervention. The increase in insulin resistance significantly correlated with an increase in the number of Ki-67 positive β-cells. Moreover, the ability to switch between fuel sources in liver tissue homogenate substrate oxidation assays revealed reduced metabolic flexibility. Furthermore, metabolomics analyses revealed a decrease in liver glycolytic metabolites, suggesting reduced glucose utilization, a finding consistent with onset of systemic insulin resistance. Physical activity was reduced, while respiratory quotient was increased, in mice receiving corticosterone. The majority of metabolic changes were reversed upon cessation of the drug regimen. Collectively, we conclude that changes in body composition and tissue level substrate metabolism are key components influencing the reductions in whole body insulin sensitivity observed during glucocorticoid administration.

Keywords: Body composition; Glucocorticoid; Insulin resistance; Metabolic flexibility; Metabolomics.

Figure 1.. Cort suppresses thioglycollate-induced recruitment of specific leukocyte populations.

Mice were placed on either cort or vehicle control following i.p. injection with 3% thioglycollate. Peritoneal exudate cells were collected 4 days later. (A) Total cell count of the peritoneal infiltrate was determined for both groups. The infiltrate was further analyzed for (B) monocytes, (C) macrophages, and (D) peritoneal B-cells/dendritic cells. Data are means ± S.E. with five mice per group. *, p ≤ 0.05.

Figure 2.. Changes in body composition, but not total body mass, appear after one week on Cort.

(A) Body mass, (B) fat mass, (C) lean mass, and (D) fluid mass were measured weekly. Dashed line indicates removal of drug/beginning of washout phase. n=8 per group. *p < 0.05. Data are represented as means ± SEM.

Figure 3.. Cort-mediated alterations in physical activity and sleep time returned to normal after drug removal.

(A-C) Activity, as measured by total number of X and Y beam breaks, (D-F) weekly food intake (g), (G-I) weekly liquid intake (g), and (J-L) weekly mean sleep time (h), in C57BL/6J mice treated with Vehicle or Cort for 1 (A, D, G J) or 2 (B, E, H, K) weeks, or following a 1 week washout (C, F, I, L). n=8 per group. *p < 0.05. Data are expressed as means ± SEM.

Figure 4.. Cort enhances RQ and Energy Expenditure.

(A-C) Mean respiratory quotient (RQ), (D-F) and mean energy expenditure (EE; kcal/h) in C57BL/6J mice treated with Vehicle or Cort for 1 (A and D; lower panel = quantification of light cycle data shown in top panels; gray bars represent lights off) or 2 (B and E; lower panel = quantification of light cycle data shown in top panels; gray bars represent lights off) weeks, or following a 1 week washout of Cort (C and F; lower panel = quantification of light cycle data shown in top panels; gray bars represent lights off). n=8 per group. Data are shown as means ± SEM.

Figure 5.. Insulin sensitivity is reduced early after Cort exposure and returns to normal during the drug washout phase.

(A-B) Insulin tolerance test (ITT) after 5 days of Vehicle (Veh) or Cort administration. (C-D) A second ITT on the same cohort of mice following 6 weeks of steroid washout. B and D show the area under the curve calculations for each respective ITT (C = 5d; D = 6w washout). (E) Serum insulin and (F) serum glucagon measured after 7 days of Veh or Cort administration (delivery) and again following a 6 week withdrawal period (washout). n=8 with data represented as means ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 6.. Cort exposure promotes markers of β-cell proliferation which correlate with insulin resistance, but not with total body mass.

(A) Triple-fluorescence staining of formalin-fixed paraffin-embedded pancreatic tissue showing insulin (green), DAPI (blue), and Ki-67 (red) in mice treated with either Vehicle or Cort for 1 week. (B) Quantification of the number of Ki-67⁺ cells per islet in C57BL/6J mice treated with Vehicle or Cort for 1 week (delivery), or following a 6 week washout of Cort (washout). (C, D) Pearson correlations of Ki-67⁺ cells per islet quantified from the 1 week delivery period (B) expressed as a function of (C) body mass, and (D) AUC (from ITT data shown in Figure 5). Veh= vehicle; AUC= area under the curve. n=8 per group. Data are expressed as means ± SEM. ****p < 0.0001.

Figure 7.. Cort alters liver metabolites and promotes hepatic metabolic inflexibility.

(A) Heatmap illustrating log₂ fold changes in relative abundance of metabolites to controls. An orange color indicates a positive fold change and a blue color indicates a negative fold change. P values are indicated with dots. n = 5 mice per group. (B) Total acyl glycerol was measured in the liver of C57BL/6J mice treated with Vehicle or Cort for 1 or 2 weeks, or following a 6 week washout of Cort (W/O). (C-D) Using 100μM [1-¹⁴C] palmitate, substrate switching was assessed by measured complete (CO₂; C) and incomplete (acid soluble metabolite; ASM; D) fat oxidation ± 1 mM pyruvate as a competing substrate. E. Complete oxidation (CO₂) of [U-¹⁴C] leucine (100 μM) was used to test effects on branch chain amino acid oxidation. C-E. Assays were run using homogenates from liver from C57BL/6J mice treated with Vehicle or Cort for 1 week (delivery; n = 8 per group), or following a 6 week washout of Cort (washout; W/O; n = 8 per group). *, p < 0.05 vehicle vs. Cort; #, p < 0.05 basal vs. pyruvate.