Lack of stress responses to long-term effects of corticosterone in Caps2 knockout mice

Chronic stress is associated with anxiety and depressive disorders, and can cause weight gain. Ca2+-dependent activator protein for secretion 2 (CAPS2) is involved in insulin release. Caps2 knockout (KO) mice exhibit decreased body weight, reduced glucose-induced insulin release, and abnormal psychiatric behaviors. We chronically administered the stress hormone corticosterone (CORT), which induces anxiety/depressive-like behavior and normally increases plasma insulin levels, via the drinking water for 10 weeks, and we examined the stress response in KO mice. Chronic CORT exposure inhibited stress-induced serum CORT elevation in wild-type (WT) mice, but not in KO mice. Poor weight gain in CORT-treated animals was observed until week 6 in WT mice, but persisted for the entire duration of the experiment in KO mice, although there is no difference in drug*genotype interaction. Among KO mice, food consumption was unchanged, while water consumption was higher, over the duration of the experiment in CORT-treated animals, compared with untreated animals. Moreover, serum insulin and leptin levels were increased in CORT-treated WT mice, but not in KO mice. Lastly, both WT and KO mice displayed anxiety/depressive-like behavior after CORT administration. These results suggest that Caps2 KO mice have altered endocrine responses to CORT administration, while maintaining CORT-induced anxiety/depressive-like behavior.

weeks ( Figure 1). We measured circulating levels of serum CORT after treatment ( Figure 2). There were no differences in basal CORT levels in the CORT-treated and untreated animals among WT and Casp2 KO mice in serum collected during the light phase (13:00-16:00) ( Figure 2a). As reported in a previous study 10 , in WT mice, serum CORT levels after forced swim stress were increased in untreated animals, but was unchanged in CORT-treated mice ( Figure 2b). However, this chronic CORT administration-induced suppression of stress-induced CORT elevation was insignificant in Caps2 KO mice ( Figure 2b) (Tukey HSD post hoc test: WT untreated vs. CORTtreated, P , 0.0003; Caps2 KO untreated vs. CORT-treated, P , 0.12). These results suggest a differential response to stress between WT and Caps2 KO mice after chronic CORT treatment.
Poor body weight gain in Caps2 KO mice is not related to food consumption during CORT treatment. During the 10-week exposure to CORT, we monitored body weight, drinking and food consumption every week. Both WT and Caps2 KO mice treated with CORT showed poor weight gain, although there is no difference in drug*genotype interaction (Two-way repeated measures ANOVA: time*genotype [F 5 2.56, P , 0.01]; time*drug [F 5 14.64, P , 0.0001]; time*genotype*drug [F 5 0.92, P , 0.52]). CORT-treated WT mice gained as much weight as untreated WT mice during the 7th week and beyond. In comparison, CORT-treated Caps2 mice displayed reduced weight gain during the duration of the experiment (One way ANOVA: *P , 0.05, **P , 0.01) ( Figure 3). Compared with the respective untreated group, water consumption in CORT-treated WT mice was increased only during the first three weeks (Figure 4a), whereas it was increased in CORT-treated Caps2 KO mice over the entire period tested (Figure 4b). Compared with the respective untreated group, food consumption in CORT-treated animals was increased during weeks 1, 2 and 4, and reduced during weeks 7, 8 and 9 in WT mice (Figure 4c), whereas it was not changed in CORT-treated Caps2 KO mice ( Figure 4d). These results suggest that chronic CORT exposure causes suppression of body weight gain in Caps2 KO mice, which is not due to a change in food consumption.
Serum insulin and leptin levels in Caps2 KO mice are not increased by chronic CORT treatment. Previous studies have shown that chronic CORT treatment causes increases in plasma insulin levels in mice [11][12][13] . We obtained serum during the light phase, and measured insulin levels by enzyme immunoassay (EIA). Similar to previous reports [11][12][13] , serum insulin levels were increased by chronic CORT treatment in WT mice ( Figure 5a). However, serum insulin levels did not increase in Caps2 KO mice after chronic CORT treatment . We also measured leptin, a hormone made in adipose tissue, which is known to fluctuate according to insulin levels and body fat 14 . We obtained serum during the light phase, and measured hormone levels by EIA. Serum leptin levels were also increased by chronic CORT treatment in WT mice ( Figure 5b). However, serum  Chronic CORT treatment in Caps2 KO mice causes anxiety/ depressive-like behavior comparable to that in WT mice. Because chronic CORT treatment mimics anxiety/depression in rodents 15 , we tested CORT-treated mice for anxiety/depressive-like behaviors. To test for anxiety-related behaviors, we performed an open field test. Mice were tested for three consecutive days, and data from the third day was used for analysis. For total center time, which reflects anxiety, CORT-treated mice showed lower values compared to untreated mice among both WT and Caps2 KO mice (Figure 6a) (Mann-Whitney U-test: WT untreated vs. WT CORT-treated, P , 0.005; Caps2 KO untreated vs. Caps2 KO CORT-treated, P , 0.006). Analysis of total distance traveled showed that there was no difference among the groups, indicating that total locomotor activity was not affected by CORT treatment (Figure 6b). To test for depressive behaviors, we performed the forced swim test. Immobility time was increased in CORT-treated mice in both WT and Caps2 KO mice (Figure 6c), indicating that CORT treatment caused similar depressive-like behavior in both genotypes (Student's t-test: WT untreated vs. WT CORTtreated, P , 0.05; Caps2 KO untreated vs. Caps2 KO CORT-treated, P , 0.02). Moreover, in an elevated plus maze test, CORT-treated Caps2 KO mouse group showed a tendency to decrease time spent in open arm compared with the other groups ( Figure S1), thereby suggesting increased anxiety in Caps2 KO mice after chronic CORT treatment.

Discussion
In this study, both WT and Caps2 KO mice were treated chronically with CORT for 10 weeks. This chronic treatment did not impact basal serum CORT levels in either WT or Caps2 KO mice. However, serum CORT levels after swim stress were increased in untreated WT mice, but not in CORT-treated WT mice, similar to results reported in a previous study 10 . Because CORT is regulated by an inhibitory feedback loop, chronic CORT administration normally suppresses the HPA axis 7 . The swim stress-induced increase in CORT levels was not suppressed in CORT-treated Caps2 KO mice. Thus, in Caps2 KO mice, the CORT inhibitory loop may be impaired.
After week 6, body weight increased similarly in both CORTtreated WT and untreated WT mice. In comparison, CORT-treated Caps2 KO mice displayed less weight gain compared with untreated    Caps2 KO mice although there is no difference in drug*genotype interaction. It is not yet clear why the difference in body weight changes between two genotypes occurred at later stage of chronic CORT treatment. Any metabolic changes after prolonged mild stress may be different between WT mice from KO mice with the loss-offunction of CAPS2-mediated insulin secretion. In addition, serum insulin and leptin levels were increased after CORT administration in WT mice as previously described [11][12][13] , but were unchanged in Caps2 KO mice. These results correspond with the roles of CORT and insulin in energy balance-CORT inhibits energy storage, causing a decrease in body weight, and insulin promotes energy storage, causing an increase in body weight 16 . Therefore, because insulin was secreted in response to CORT administration in WT mice, body weight increased similarly to that in untreated mice. However, because insulin was not increased in Caps2 KO mice, CORT administration caused poor weight gain in these animals.
We also conducted tests that evaluate anxiety/depressive behavior. Despite the defects in insulin and leptin release during CORT treatment, Caps2 KO mice did not exhibit heightened anxiety/depressive behavior compared with WT mice. This result suggests that chronic CORT administration does not exacerbate anxiety/depression in Caps2 KO mice.
Studies have shown that symptoms in adrenalectomized rats, which include reduced caloric intake and poor weight gain, are corrected by sucrose drink or intraventricular insulin and leptin administration [17][18][19] . These studies suggest that there is a metabolic feedback system that suppresses stress responses. However, the underlying regulatory mechanisms are unclear. CORT-treated Caps2 KO mice, which lack increased insulin release, may help to clarify the roles of metabolic feedback in stress responses.
Collectively, the findings of the present study show that chronic CORT treatment causes differential endocrine responses in Caps2 KO and WT mice. These differences in endocrine responses, however, were not associated with differences in anxiety/depressive behavior. This suggests that weight gain as a response to stress does not substantially impact anxiety-related stress. Most studies examining the roles of CORT, insulin and leptin in obesity and behavior have used adrenalectomized rats, which require complicated surgery. Thus, the CORT-treated Caps2 KO mice used in the present study may serve as a useful model for investigating the mechanisms of CORT and insulin in depression and obesity.

Methods
Animals. Caps2 KO mice 5,6 and WT littermate controls (background: C57BL/6J) were used for the study. Male mice (6-8-week-old) were housed four per cage (according to genotype) and maintained under a 12512 light-dark cycle (lights on at 08:00). The experiments were approved by the Institutional Animal Care and Use Committee of RIKEN and Tokyo University of Science. All experiments were conducted in accordance with the Regulations for Animal Research at the RIKEN and Tokyo University of Science.
Drugs. CORT (Sigma, St. Louis, MO) was dissolved in ethanol (EtOH) and diluted in tap water at a concentration of 25 mg/ml for WT mice and 22 mg/ml for Caps2 KO mice (final EtOH concentration of 0.2% for WT mice and 0.18% for Caps2 KO mice). The concentration of CORT was adjusted according to the weight of each genotype. CORT or EtOH control was delivered in the drinking water, and solutions were replaced every 2-3 days.
Experimental design. Both WT and Caps2 KO mice were divided into two groups; one group was treated with EtOH as a control (untreated group) and the other group was treated with CORT (CORT-treated group). Male mice were 7-8 weeks old at the start of the experiment. The experiment was repeated twice, and data were combined for behavioral testing. For the first set of mice, WT EtOH group, n 5 12, WT CORT group, n 5 12, Caps2 KO EtOH group, n 5 8, Caps2 KO CORT group, n 5 12. For the second set, WT EtOH group, n 5 8, WT CORT group, n 5 8, Caps2 KO EtOH group, n 5 6, Caps2 KO CORT group, n 5 6. Body weight, food consumption and drinking volume were measured once per week. After 7 weeks of CORT treatment, the open field test (days 48, 49, 50 or days 49, 50, 51) and the forced swim test (between days 55 and 59) were conducted. After 10 weeks, mice were sacrificed, and brain regions and serum were collected (Figure 1).
Behavioral tests. Behavioral tests were performed between 13:00 and 17:00. Mice were acclimated to the behavioral room at least 1 h before testing.
Open field test. Mice were tested in the open field for 15 min per session for 3 consecutive days according to a previously published protocol 20 . The design permits assessment of exploratory behavior on days 1 and 2, and anxiety on day 3, because by day 3, mice are habituated to the open field. The open field test was performed as previously described 6 . Briefly, locomotor activity was measured with a light level of 70 lux in an open field apparatus (60 cm 3 60 cm). Each mouse was placed at the left corner of the open field, and horizontal movements were recorded using Image J OF software (O'Hara & Co., Ltd., Tokyo, Japan). Total activity for 15 min on the third day was used for statistical analysis.
Forced swim test. The forced swim test was performed as previously described 21 . Briefly, mice were placed into plastic buckets, 19 cm in diameter and 23 cm deep, filled with water at 25uC, and mobility was recorded for 6 min. The last 5 min were scored for immobility. Immobility duration was scored using FST software (O'Hara & Co., Ltd.).
Enzyme immunoassay of serum corticosterone, insulin and leptin. On week 8, blood was collected by tail incision after the forced swim test. On week 10, mice were decapitated, and trunk blood was collected during the light phase (13:00-16:00). Blood was collected in 1.5-ml plastic tubes and kept at 4uC overnight. Blood was centrifuged at 1,000 3 g for 20 min, and serum was removed and stored at 280uC until use. Serum CORT was measured using an enzyme immunoassay kit (Assay Designs; Enzo Life Sciences, Farmingdale, NY). Serum insulin and leptin were measured using enzyme immunoassay kits (Millipore, Billerica, MA) (n 5 6-8 for each group). Blood was also collected during the dark phase (20:00-22:00) on week 9 of CORT administration, and in the morning (09:00-11:00) on week 10, 3-4 hours before decapitation (data not shown).
Statistical analysis. Data were analyzed using JMP software (SAS Institute, Cary, NC) and Excel (Microsoft, Redmond, WA). Data were analyzed by one-way, two-way or repeated measures ANOVA, followed by Tukey HSD post hoc test or Student's t-test. Values in graphs are expressed as mean 6 SEM.