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Age- and gender-related changes in glucose homeostasis in glucocorticoid-treated rats Cristiane dos Santos, Francielle Batista D. Ferreira, Luiz M. Gonçalves-Neto, Sebastião Roberto Taboga, Antonio Carlos Boschero, and Alex Rafacho

Abstract: The disruption to glucose homeostasis upon glucocorticoid (GC) treatment in adult male rats has not been fully characterized in older rats or in females. Thus, we evaluated the age- and gender-related changes in glucose homeostasis in GC-treated rats. We injected male and female rats at 3 months and 12 months of age with either dexamethasone (1.0 mg/kg body mass, intraperitoneally) or saline, daily for 5 days. All of the GC-treated rats had decreased body mass and food intake, and adrenal hypotrophy. Increased glycemia was observed in all of the GC-treated groups and only the 3-month-old female rats were not glucose intolerant. Dexamethasone treatment resulted in hyperinsulinemia and hypertriacylglyceridemia in all of the GC-treated rats. The glucose-stimulated insulin secretion (GSIS) was higher in all of the dexamethasone-treated animals, but it was less pronounced in the older animals. The ␤-cell mass was increased in the younger male rats treated with dexamethasone. We conclude that dexamethasone treatment induces glucose intolerance in both the 3- and 12-month-old male rats as well as hyperinsulinemia and augmented GSIS. Three-month-old female rats are protected from glucose intolerance caused by GC, whereas 12-month-old female rats developed the same complications that were present in 3- and 12-month-old male rats. Key words: dexamethasone, female, glucose tolerance, insulin secretion, insulin sensitivity. Résumé : La perturbation de l'homéostasie du glucose a` la suite d'un traitement aux glucocorticoïdes (GC) observée chez des rats adultes mâles n'a pas encore été pleinement caractérisée chez les rats plus âgés et chez les femelles. Ainsi, les auteurs ont évalué les changements dans l'homéostasie du glucose reliés a` l'âge et au genre chez des rats traités aux GC. Ils ont injectés des rats mâles et femelles de 3 et 12 mois, soit avec de la dexaméthasone (1.0 mg/kg de poids corporel, par voie intra-péritonéale) ou de la saline pendant 5 jours. Tous les rats traités aux GC présentaient une diminution du poids corporel et de la prise alimentaire ainsi qu'une hypotrophie surrénalienne. Une élévation de la glycémie a été observée chez tous les rats traités aux GC, et seules les femelles de 3 mois n'étaient pas intolérantes au glucose. Le traitement a` la dexaméthasone résultait en une hyperinsulinémie et une hypertriglycéridémie chez tous les rats. La sécrétion d'insuline stimulée par le glucose (SISG) était plus élevée chez tous les animaux traités a` la dexaméthasone, mais elle était moins prononcée chez les animaux plus âgés. Globalement, les auteurs concluent que le traitement a` la dexaméthasone induit une intolérance au glucose chez les rats mâles de 3 et 12 mois, ainsi qu'une hyperinsulinémie et une SISG accrue. Les femelles de 3 mois sont protégées de l'intolérance au glucose induite par les GC, alors que les femelles de 12 mois développent les mêmes complications que les mâles de 3 et 12 mois. [Traduit par la Rédaction] Mots-clés : dexaméthasone, femelle, tolérance au glucose, sécrétion d'insuline, sensibilité a` l'insuline.

Introduction Glucocorticoids (GCs) are widely used in clinical practice because of their desirable anti-inflammatory and immunosuppressive properties. However, excessive endogenous or exogenous levels of GCs may cause an imbalance in overall metabolism, especially with respect to glucose metabolism (Schäcke et al. 2002; Gathercole et al. 2013). The adverse effects of GCs on glucose homeostasis are well known in human subjects (Beard et al. 1984; Willi et al. 2002; Nicod et al. 2003) and have been well reproduced in rodent models subjected to GC treatment (Stojanovska et al. 1990; Novelli et al. 1999; Rafacho et al. 2010, 2011). The side effects of GC on rat glucose homeostasis may include increased blood glucose levels, glucose intolerance, reduction of insulin sensitivity and (or) glucose clearance, and the presence of hyperinsulinemia

(for a review see Rafacho et al. 2012). When GC is administered under specific conditions, e.g., to obese rats (Ohneda et al. 1993) or rats prone to develop diabetes (Ogawa et al. 1992), it may aggravate an already disrupted glucose metabolism and lead to the development of overt hyperglycemia. These vulnerable conditions reinforce the importance of understanding the impact of GC exposure in different contexts (e.g., during pregnancy, aging). Furthermore, the increased glucose-stimulated insulin secretion (GSIS) (Karlsson et al. 2001; Giozzet et al. 2008; Rafacho et al. 2008a) and pancreatic ␤ cell proliferation (Choi et al. 2006; Rafacho et al. 2010, 2011) after exposure to GCs has contributed to the understanding of compensatory adaptations that occur in rats in an attempt to avoid an imbalance in glucose homeostasis. However, whether these adaptive compensations in the endocrine

Received 9 July 2014. Accepted 26 August 2014. C. dos Santos,* F.B.D. Ferreira,* L.M. Gonçalves-Neto, and A. Rafacho. Department of Physiological Sciences, Center of Biological Sciences, Federal University of Santa Catarina (UFSC), 88040-900 Florianópolis, Brazil. S.R. Taboga. Department of Biology, Institute of Biosciences, Humanities and Exact Sciences, São Paulo State University (UNESP), São José do Rio Preto, Brazil. A.C. Boschero. Department of Structural and Functional Biology, Institute of Biology, and Obesity and Comorbidities Research Center (OCRC), State University of Campinas (UNICAMP), Campinas, Brazil. Corresponding author: Alex Rafacho (e-mail: [email protected]). *These authors contributed equally to this work. Can. J. Physiol. Pharmacol. 92: 867–878 (2014) dx.doi.org/10.1139/cjpp-2014-0259

Published at www.nrcresearchpress.com/cjpp on 27 August 2014.

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pancreas to GCs occur in a similar manner in females and males, and especially in aging rats, has not been fully investigated. Aging is a natural process that increases the vulnerability of glucose metabolism. There is evidence that the glucose homeostasis of elderly men and women can mildly deteriorate towards an increase in basal glucose concentration and the emergence of glucose intolerance (Palmer and Ensinck 1975; Fink et al. 1984; Pacini et al. 1988; Shimizu et al. 1996; Song et al. 2007; Lin et al. 2011). The reduction of insulin sensitivity owing to inactivity and (or) increased adiposity (Pacini et al. 1988) and an increase in the plasma proinsulin to insulin ratio (Shimizu et al. 1996; Gama et al. 2000) are some of the mechanisms ascribed for such glucose dysregulation. Little is known regarding glucose homeostasis in aging rats, but there is evidence that relatively poor insulin secretion could be one of the mechanisms that cause these animals to be more prone to glucose intolerance (Reaven et al. 1983; Novelli et al. 1999). Much less is known regarding the effects of GCs on glucose homeostasis in older rats. One report with 24-month-old male rats reported that 7 continuous days of dexamethasone treatment resulted in anorexia that was associated with hyperleptinemia and hyperinsulinemia (Caldefie-Chézet et al. 2001). Another group demonstrated that continuous dexamethasone administration to male rats for 13 days caused hyperinsulinemia in young and old rats, but hyperglycemia was only observed in the older rats, which could be due to insufficient compensatory insulin secretion from pancreatic ␤-cells (Novelli et al. 1999). Our knowledge on gender differences in rat glucose homeostasis is scarce; however, it appears that normal female rats exhibit higher basal plasma insulin values compared with the male rats (Vital et al. 2006; De Toro-Martín et al. 2014). The main changes observed for insulin sensitivity and glucose tolerance in the healthy adult subjects treated with dexamethasone for up to 4 days (with doses varying from 2 to 6 mg per day) (Beard et al. 1984; Willi et al. 2002; Nicod et al. 2003) are also observed in the adult rats receiving dexamethasone for up to 12 days (with doses varying from 1 to 2 mg/kg body mass) (Karlsson et al. 2001; Giozzet et al. 2008; Rafacho et al. 2008a, 2010, 2011). To the best of our knowledge, there is no study conducted in rats that evaluated age- and gender-related changes in glucose homeostasis caused by GC treatment. This question merits investigation because both age and gender may lead to different GC effectiveness, which is important for clinical applications. It was previously demonstrated in dose(Rafacho et al. 2008a) and time-dependent studies (Rafacho et al. 2011) that adult male rats treated with 1.0 mg/kg body mass dexamethasone for 5 consecutive days show symptoms that mimic the metabolic aspects of prediabetes (hyperinsulinemia, glucose intolerance, decreased insulin sensitivity, and increased ␤-cell mass). By treating 3-month-old and 12-month-old male and female rats with dexamethasone for 5 consecutive days, we demonstrated that adult female rats are protected from the consequences of dexamethasone on glucose tolerance. However, we demonstrated that older female rats reproduce all of the signs of dysregulation (glucose intolerance, dysglycemia, dyslipidemia, insulin resistance) that were observed in the 3- and 12-month-old male rats.

Materials and methods Animals and ethical approval Experiments were performed with 3-month-old and 12-monthold male and female Wistar rats. The rats were obtained from the animal breeding center at the Federal University of Santa Catarina. They were kept at 21 ± 1 °C on a 12 h (light) – 12 h (dark) cycle (lights on at 0600 h – lights off at 1800 h) and had access to commercial standard chow and water ad libitum. According to national regulations, the experiments were approved by the Committee for Ethics in Animal Experimentation at the Federal University of Santa Catarina (approval ID: PP00599) and in accordance with the Guide for the Care and Use of Laboratory Animals (NAC 2011).

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Materials Dexamethasone phosphate (Decadron) was obtained from Aché (Campinas, São Paulo, Brazil). Human recombinant insulin (Humulin) was acquired from Lilly (Indianapolis, Indiana, USA). The reagents used for the insulin radioimmunoassay (RIA) to determine hepatic glycogen and pancreas histology were purchased from LabSynth (Diadema, São Paulo, Brazil) and Sigma (St. Louis, Missouri, USA). The 125I-labeled insulin (human recombinant) for the RIA was purchased from PerkinElmer (Waltham, Massachusetts, USA). Dexamethasone treatment and serum measurements Rats in the dexamethasone treatment group received a single intraperitoneal (i.p.) injection (0730–0830 h) of dexamethasone (1 mg/kg body mass (b.m.)) daily, for 5 consecutive days, as previously described (Caperuto et al. 2006; Rafacho et al. 2007; De Paula et al. 2011). Rats in the control group (CTL) received saline (1 mL of 0.9% NaCl/kg b.m.). The animals were distributed among 8 groups as follows: 3-month-old male saline- (MC3) or dexamethasone-treated (MD3) rats; 12-month-old male saline(MC12) or dexamethasone-treated (MD12) rats; 3-month-old female saline- (FC3) or dexamethasone-treated (FD3) rats; and 12-month-old female saline- (FC12) or dexamethasone-treated (FD12) rats. Body mass and food intake were measured 2 days prior to the beginning of the injections, and were observed daily until the rats were euthanized. Fasted (12–14 h) rats had blood collected from the tail to measure the blood glucose levels with a glucometer (Accu-Check; Performa, Roche Diagnostics GmbH, Mannhein, Germany). Immediately after this, they were euthanized (exposure to CO2 followed by decapitation), and the trunk blood was collected to obtain serum. After the serum was collected, the blood was clotted and centrifuged in saline-washed tubes, and then the insulin levels were measured using RIA with a guinea pig anti-rat insulin antibody and rat insulin as the standard (Rafacho et al. 2010). Adrenal glands (left and right) were resected immediately after sacrifice, cleared of fat, weighed, and photographed. Adrenal hypotrophy after dexamethasone treatment for 5 consecutive days (1 mg/kg b.m.) is significantly correlated with reduced serum corticosterone levels in this rat model, as demonstrated previously (Rafacho et al. 2008a). We used adrenal mass as a surrogate measure of dexamethasone activity. The total cholesterol (T-cholesterol) and triacylglycerol concentrations were analyzed with enzymatic colorimetric assays (Labtest, Lagoa Santa, Minas Gerais, Brazil) according to the manufacturer’s instructions, using a spectrophotometer (Berthold Technologies, Bad Wildbad, Germany). Liver glycogen and fat measurements The hepatic glycogen content was determined as previously described (Rafacho et al. 2007). Briefly, liver samples (300 to 500 mg) were transferred to test tubes containing 30% KOH (w/v) and boiled for 1 h until completely digested. Then, Na2SO4 was added and the glycogen was precipitated with ethanol. The samples were centrifuged at 800g for 10 min, the supernatants were discarded, and the glycogen was dissolved in hot distilled water. Ethanol was added and the pellets that were obtained after a second centrifugation were dissolved in distilled water to a final volume of 20 mL. The glycogen content was measured by treating a fixed volume of the sample with a phenol reagent and H2SO4, and the absorbance was read at 490 nm with a spectrophotometer. For the determination of hepatic fat content, liver samples (100 mg) were transferred to test tubes containing 0.7 mL of 1 mol/L NaCl and homogenized with T18 UltraTurrax (IKA; Staufen, Germany). Then, 2 mL of methanol–chloroform solution (1:2 v/v) were added and the tubes were subsequently centrifuged for 5 min at 800g. The methanolic phase was then transferred to another test tube and dried under N2, then a solution of methanol–Triton 100 Published by NRC Research Press

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Fig. 1. Body mass and food intake. The average body mass during dexamethasone treatment in 3- and 12-month-old male (A) and female (C) rats. There was a significant reduction in body mass in all of the GC-treated groups on the fifth day of dexamethasone treatment compared with the control groups. The average food intake values during dexamethasone treatment in 3- and 12-month-old male (B) and female (D) rats. There was a significant reduction in food intake in all of the GC-treated groups on the fifth day of dexamethasone treatment compared with the control groups. The values are the mean ± SEM of 8 rats per group; *, p < 0.05 compared with the respective control group (effect of dexamethasone); #, p < 0.05 compared with the respective control group (effect of age). The data were analyzed by either one-way ANOVA followed with a Tukey post-hoc test, or by Kruskal–Wallis non-parametric ANOVA followed with a Dunn post-hoc test.

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glucose values between 0 and 16 min after insulin administration (linear phase of glucose decay) (Rafacho et al. 2007, 2010).

Intraperitoneal glucose tolerance test (ipGTT) Separate groups of fasted (12–14 h) rats were used for the ipGTT experiments. Blood samples were collected from conscious rats and were used for measuring glycemia (time 0) using a glucometer, as described above. Immediately following this, a 50% glucose solution (2 g/kg b.m., i.p.) was administered and blood samples were collected from the tail tip at 30, 60, and 120 min to measure the blood glucose levels (Rafacho et al. 2007, 2010).

Isolation of islet and glucose-stimulated insulin secretion Islets were isolated by collagenase digestion of the pancreas with minor modifications, as described previously (Rafacho et al. 2008a). For static incubation, groups of 5 islets were first incubated for 1 h at 37 °C in 1 mL Krebs–Ringer bicarbonate buffer solution of the following composition (in mmol/L): 115 NaCl, 5 KCl, 2.56 CaCl2, 1 MgCl2, 24 NaHCO3, 15 HEPES, and 5.6 glucose, supplemented with 0.1% of bovine serum albumin and equilibrated with a mixture of 95% O2 : 5% CO2, pH 7.4. The medium was then replaced with fresh buffer containing 5.6 or 16.7 mmol/L glucose and further incubated for 1 h. At the end of the incubation the samples were stored at –20 °C for subsequent measurement of insulin content by AlphaLISA system according to the manufacturer's instructions, using the Enspire Alpha reader (Perkin–Elmer).

Intraperitoneal insulin tolerance test (ipITT) Separate groups of fasted (12–14 h) rats were used for the ipITT experiments. Conscious rats had their tail tips cut to collect blood. The first drop was discarded and the second was used to determine glycemia (time 0) using a glucometer as described above. Immediately, human recombinant insulin (equivalent to 2 IU/kg b.m., i.p.) was administered. Additional blood samples were collected at 8, 16, and 24 min to measure the blood glucose levels. The constant rate for glucose disappearance (KITT) was calculated from the slope of the regression line obtained with log-transformed

Quantitative morphometric analysis of the endocrine pancreas To study the morphological aspects and pancreatic ␤-cell mass of the pancreas, pancreases from 5 rats from each group were Published by NRC Research Press

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Fig. 2. (A and C) Representative adrenal glands photographed immediately after sacrifice. Note the visible hypotrophy in both adrenals from the dexamethasone-treated rats. (B and D) The columns illustrate the gland mass for all of the groups. The values are the mean ± SEM; n = 16 glands from 8 animals; *, p < 0.05 compared with the respective control group (effect of dexamethasone); #, p < 0.05 compared with the respective control group (effect of age). The data were analyzed using one-way ANOVA followed by a Tukey post-hoc test. Scale bar = 10 mm.

excised cleared of fat and lymph nodes, and weighed. Only the splenic portions of the pancreas were immersion-fixed for 12 h in Bouin's fixative solution, dehydrated, and embedded in paraffin according to the methods of Rafacho et al. (2008b). The largest pancreas area from the block was cut (5 ␮m) on a rotary microtome and adhered to individual glass slides. The section was then insulin immunostained for morphological and stereological analysis as previously described (Rafacho et al. 2008b, 2011). The ␤-cell mass was determined by point counting stereology as previously described (Rafacho et al. 2008b, 2011), with minor modifications. Briefly, each section was systematically scored with a grid of 196 points (final magnification 200×) using ImageJ software version 1.45S. The numbers of intercepts over ␤-cells, non-␤-cells, exocrine tissue, and non-exocrine tissue were counted. The ␤-cell relative volume was calculated by dividing the intercepts over ␤-cells by the intercepts over the total pancreatic tissue; the absolute ␤-cell mass was then estimated by multiplying the ␤-cell relative volume by the total pancreas mass. A total of 2109 (MC3), 2495 (MD3), 2252 (MC12), 2587 (MD12), 1828 (FC3), 2077 (FD3), 1677 (FC12), and 1709 (FD12) fields was counted for the pancreases from each group. Data analysis The values for the results are the mean ± SEM of the indicated number (n) of the experiments. We used one-way analysis of variance for unpaired groups followed by Tukey's post-hoc test for multiple comparisons of parametric data. When necessary, the nonparametric Kruskal–Wallis test followed by the Dunn posthoc test was applied. When text refers to “their respective control groups”, this denotes MD3 compared with MC3, MD12 compared

with MC12, FD3 compared with FC3, or FD12 compared with FC12. Values for p < 0.05 were considered statistically significant.

Results Body mass, food intake, and adrenal mass Treating adult male rats (approximately 3 months old) with dexamethasone is known to induce a significant reduction in both body mass and food intake during treatment (Caperuto et al. 2006; Rafacho et al. 2007). Our experiments clearly demonstrated that dexamethasone induced a significant reduction in body mass for all of the groups studied compared with their controls (Figs. 1A and 1C; p < 0.05, n = 8), suggesting that there were no age- and (or) gender-related differences among them. There were no differences in the initial body mass of the control or the dexamethasonetreated groups before GC treatment. The magnitude of reduction in body mass on the day after the final dexamethasone administration was 15% (MD3), 10% (MD12), 11% (FD3), and 8% (FD12) compared with their respective controls. The food intake values before and during dexamethasone administration were lower in the 12-month-old compared with the 3-month-old saline-treated rats for both genders (Figs. 1B and 1D; n = 8, p < 0.05). The reduction in the food intake was obvious on the third day of dexamethasone administration for the MD3, MD12, and FC3 groups, but this reduction was not apparent in the FD12 rats until the fifth day (p < 0.05). Figure 2A shows representative adrenal glands and the respective mean mass. The MC12 and FC12 rats exhibited a significant reduction in adrenal mass compared with the MC3 and FC3 rats, respectively (Figs. 2B and 2D; p < 0.05, n = 16 glands from 8 animals). The adrenal gland hypotrophy was more pronounced Published by NRC Research Press

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Fig. 3. Blood glucose and serum insulin. Blood glucose levels in 3- and 12-month-old (A) male and (C) female rats. The fasting blood glucose values were significantly increased in both male and female groups of glucocorticoid (GC)-treated rats. Serum insulin levels in the 3- and 12-month-old (B) male and (D) female rats. Fasting serum insulin values were significantly increased in all of the GC-treated groups compared with their respective controls. The values are the mean ± SEM; n = 14 rats for A and C, and 8 rats for B and D; *, p < 0.05 compared with the respective control group (effect of dexamethasone). The data were analyzed using either one-way ANOVA followed by a Tukey post-hoc test, or with a Kruskal–Wallis non-parametric ANOVA followed by a Dunn post-hoc test.

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in the GC-treated animals, with a mass reduction of 37% for the MD3, 30% for the MD12, 31% for the FD3, and 20% for the FD12 group compared with their respective controls (p < 0.05). These data strengthen the anorexic effects of GCs when administered in excess for prolonged periods. Blood glucose, serum insulin, and hepatic glycogen content The impact of GCs on the fasting glycemic values is known to be either absent or negative depending on dosing regimen and the period of GC administration (Novelli et al. 1999; Rafacho et al. 2008a). No differences regarding the fasting blood glucose and serum insulin values were observed between the 3-month- and 12-month-old saline-treated rats of either gender (Figs. 3A–3D; p < 0.05, n = 8–14), suggesting that there was no discernible influence of either age or gender on basal glucose homeostasis. However, the blood glucose levels were augmented in the MD3 (21%), MD12 (40%), FD3 (23%), and FD12 (43%) rats compared with the MC3, MC12, FC3, and FC12 rats, respectively (Figs. 3A and 3C; n = 14, p < 0.05). The serum insulin values at the end of dexamethasone administration were markedly increased in all of the groups (MD3, MD12, FD3, and FD12) (Figs. 3B and 3D; n = 8, p < 0.05). The magnitudes of the increase were 7.7-fold (MD3), 9.7-fold (MD12), 6.7-fold (FD3), and 6.8-fold (FD12) compared with their respective

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controls. The hepatic glycogen content (mg/100 mg of tissue) was enhanced in all of the dexamethasone-treated rats: 1.1 ± 0.1 for MC3, 5.4 ± 0.2 for MD3, 0.4 ± 0.1 for MC12, 3.7 ± 0.1 for MD12, 0.2 ± 0.05 for FC3, 2.7 ± 0.4 for FD3, 0.2 ± 0.05 for FC12, and 4.4 ± 0.3 for FD12 (n = 8, p < 0.05). These data reinforce the negative impact of GCs on glucose homeostasis, which causes compensatory hyperinsulinemia that is apparent in all of the groups, independent of gender and age. Glucose tolerance and insulin sensitivity GCs are known to induce glucose intolerance in healthy adult men (Nicod et al. 2003) as well as in adult male rats (Giozzet et al. 2008; Rafacho et al. 2011). However, there were no comparative studies that evaluated age- and gender-related changes in glucose tolerance at the same time. Figures 4A and 4C show the blood glucose concentrations from the ipGTT assay in both male and female rats, respectively (n = 8). No significant increase in the blood glucose values was observed at 30, 60, and 120 min in the MC12 rats compared with the MC3 rats after the intraperitoneal glucose loading. No apparent changes in the blood glucose values were observed between FC12 and FC3 rats and between the FD3 and FC3 rats during the ipGTT experiment. As expected, dexamethasone treatment resulted in glucose intolerance in the MD3 Published by NRC Research Press

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Fig. 4. Glucose tolerance. (A and C) The average blood glucose values during an intraperitoneal glucose tolerance test in 3- and 12-month-old male and female rats, respectively. Dexamethasone treatment resulted in glucose intolerance both in the MD3 and MD12 rats as well as in the FD12 rats compared with their respective controls. The area-under-the-curve for glucose values (B and D) confirms the glucose intolerance for the aforementioned groups. The values are the mean ± SEM from 8 rats per group; *, p < 0.05 compared with the respective control group (effect of dexamethasone); #, p < 0.05 compared with the respective control group (effect of age). The data were analyzed using either one-way ANOVA followed by a Tukey post-hoc test, or by Kruskal–Wallis non-parametric ANOVA followed by a Dunn post-hoc test.

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compared with the MC3 rats, as observed at 60 and 120 min after the glucose challenge and the area-under-the-curve (AUC) data (Figs. 4A and 4B; p < 0.05). The older male rats treated with dexamethasone had a similar glycemic profile to that of the MD3 rats, as can be observed from the AUC data. The older female rats exhibited significant glucose intolerance after dexamethasone treatment (Figs. 4C and 4D; n = 8). Taken together, it appears that the 3-month-old female rats were protected from the effects of GC on glucose tolerance but became vulnerable to these effects as they aged. To confirm whether this glucose intolerance is accompanied by a reduction in insulin sensitivity, we performed ipITT experiments (Figs. 5A–5D). Older male rats (MC12) had no apparent alteration in insulin sensitivity compared with the MC3 rats, as determined from the KITT values. However, dexamethasone treatment resulted in decreased insulin sensitivity in the MD12 rats, compared with the MC12 rats (Figs. 5A and 5B; n = 8, p < 0.05). The AUC data were 15% and 27% higher in the MD3 and MD12 rats, respectively, compared with the MC3 and MC12 rats (n = 8, p < 0.05; data not shown), corroborating the KITT results. Twelvemonth-old female rats presented with decreased insulin sensitivity compared with the FC3 rats (Figs. 5C–5D; n = 8, p < 0.05). Three-month-old female rats (FD3) were partially protected from

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the GC-induced reduction in insulin sensitivity, and 12-month-old female rats had no additive effect of dexamethasone on insulin insensitivity caused by age (FD12) (Figs. 5C and 5D). These data reinforce the negative impact of GC on male insulin sensitivity and the female resistance to such effect. Insulin secretion in response to glucose Since glucose intolerance may be related to impaired GSIS (Weir and Bonner-Weir 2004), we next evaluated insulin secretion in response to basal (5.6 mmol/L) and stimulatory (16.7 mmol/L) glucose concentrations. Incubation of islets isolated from the MD3 and MD12 rats had enhanced insulin response at basal and stimulatory glucose concentrations compared with the MC3 and MC12 rats, respectively (Fig. 6A; n = 6 wells, 2 rats; p < 0.05). GSIS from islets of the MD12 rats were lower (22%) than that of the MD3 rats (p < 0.05). As for the male rats, islets from the female rats treated with dexamethasone (FD3 and FD12) had augmented insulin release in response to both 5.6 and 16.7 mmol/L glucose (Fig. 6B; n = 6 wells, 2 rats; p < 0.05). The FD12 rats also had lower GSIS (42%) compared with the FD3 rats (p < 0.05). GSIS from islets of the FC12 rats was 55% of that in the FC3 rats (p < 0.05), which is in accordance with their glucose intolerance. These data indicate Published by NRC Research Press

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Fig. 5. Insulin sensitivity. (A and C) The average blood glucose values during an intraperitoneal insulin tolerance test in 3- and 12-month-old male and female rats, respectively. Dexamethasone treatment attenuated the insulin sensitivity in 12-month-old GC-treated male rats as well as in both 12-month female groups compared with their respective controls, as validated by the change (disappearance) in the constant (KITT value) for glucose (B and D). The values are the mean ± SEM from 8 rats per group; *, p < 0.05 compared with the respective control group (effect of dexamethasone); #, p < 0.05 compared with the respective control group (effect of age). The data were analyzed using either one-way ANOVA followed by a Tukey post-hoc test, or Kruskal–Wallis non-parametric ANOVA followed by a Dunn post-hoc test.

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the negative impact of age on endocrine pancreas ability to properly respond to different glucose concentrations. Serum triacylglycerol and T-cholesterol, and hepatic fat content Because an imbalance in glucose homeostasis may be followed by an imbalance in lipid homeostasis (Novelli et al. 1999; Rafacho et al. 2008a), we assessed the basic lipid components in the sera from all of the groups. No differences were observed between the MC12 and MC3 rats, or between the FC12 and FC3 rats with regard to fasting serum triacylglycerol and T-cholesterol levels (Figs. 7A– 7D; n = 8), suggesting no effect of aging on these variables. All of the dexamethasone-treated groups had an apparent increase in fasting triacylglyceridemia (Figs. 7A and 7D; p < 0.05). The magnitude of the increases were 157% (MD3), 260% (MD12), 76% (FD3), and 97% (FD12) compared with the MC3, MC12, FC3, and FC12 rats, respectively. The circulating T-cholesterol levels were similar among the groups (Figs. 7B and 7E). The hepatic triacylglycerol content was similar between the MC3 and MC12 rats, but increased markedly after dexamethasone treatment in the liver of the MD12 rats (Fig. 7C; n = 6 fragments, 2 rats; p < 0.05). The hepatic triacylglycerol contents were similar between the FC3 and FC12 rats, but were significantly elevated in the FD3 and

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FD12 rats compared with their respective controls (Fig. 7F; n = 6 fragments, 2 rats; p < 0.05). These data reinforce the negative impact of GCs on triacylglycerol metabolism. Pancreatic ␤-cell mass Considering that the reduction in insulin sensitivity is reciprocally compensated by augmented ␤-cell mass (Kahn et al. 2006), we investigated the pancreas from the rats in all of our groups. Figures 8A and 8B represent a panoramic view of the pancreas sections immunostained to insulin for all of the groups. No significant alterations were observed with regards to the exocrine tissue among all of the groups. The average mass of the pancreas was as follows: 956 ± 62 mg for MC3, 837 ± 25 mg for MD3, 958 ± 45 mg for MC12, 916 ± 64 mg for MD12, 722 ± 82 mg for FC3, 566 ± 26 mg for FD3, 790 ± 89 mg for FC12, and 634 ± 33 for FD12. Twelvemonth-old male rats exhibited some degree of islet hypertrophy, but did not have expanded ␤-cell mass (Figs. 8A, B; n = 5). Dexamethasone-treated rats (MD3 and MD12) had discernible islet hypertrophy that was parallel to a 95% (MD3) and 57% (MD12) increase in their absolute ␤-cell mass, respectively, compared with the MC3 and MC12 rats, respectively (Figs. 6A and 6B; p < 0.05, only for MD3). No significant changes were evident between the ␤-cell mass for FC12 and FC3 (Fig. 8C). Although no statistically Published by NRC Research Press

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Fig. 6. Glucose-induced insulin release and sensitivity. Insulin release from isolated islets in 3- and 12-month-old (A) male and (B) female rats. At the concentration of 2.8 mmol/L glucose, insulin secretion was higher in all of the glucocorticoid (GC)-treated groups. At a concentration of 16.7 mmol/L glucose, insulin secretion was higher in all of the GC-treated groups, but of lower magnitude in the older rats, independent of gender. The values are the mean ± SEM; n = 6 wells, 2 rats; a, p < 0.05 compared with the respective basal condition (2.8 mmol/L); *, p < 0.05 compared with the respective control group (effect of dexamethasone); #, p < 0.05 compared with the respective control group (effect of age). The data were analyzed using either one-way ANOVA followed by a Tukey post-hoc test, or Kruskal–Wallis non-parametric ANOVA followed by a Dunn post-hoc test.

significant differences were observed regarding the ␤-cell mass data (Fig. 8D, n = 5, NS), both groups of dexamethasone-treated (FD3 and FD12) rats presented hypertrophied islets (Fig. 8C). These data suggest that male rats are more prone to the metabolic adverse effects of GCs, which requires a compensatory increase of ␤-cell mass.

Discussion The classical adverse effects of GC therapy on glucose homeostasis have been well-known for decades. These effects are well characterized in both humans (Willi et al. 2002; Nicod et al. 2003; Binnert et al. 2004) and rats (Novelli et al. 1999; Caperuto et al. 2006; Angelini et al. 2010; Rafacho et al. 2011) and include in-

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creased glycemia, hyperinsulinemia, dyslipidemia, reduction of peripheral insulin sensitivity, glucose intolerance, and alterations in pancreatic ␤-cells. However, it is unknown whether these adverse effects of GCs on rat glucose homeostasis occur in a similar manner, when considering age and gender. Here, we show that dexamethasone treatment for 5 consecutive days exerts anorexic actions, causes adrenal hypotrophy, and induces hyperinsulinemia and hypertriacylglyceridemia in all of the groups that receive the GC. The 3-month-old female rats are protected from the glucose intolerance that is observed in the older female rats and in both male groups after GC treatment. We also show that GSIS are elevated in all of the dexamethasone-treated rats, independent of age and gender, but it is less pronounced in islets of the older animals. Also, GC results in increased pancreatic ␤-cell mass only in the 3-month-old male rats. Reductions in body mass and food intake in the adult male rats have been previously demonstrated by our group and others (Caperuto et al. 2006; Giozzet et al. 2008; Rafacho et al. 2010, 2011). The effect of dexamethasone in this study was neither age- nor gender-related, because all of the groups receiving GCs exhibited reduced body mass during the treatment. This reduced body mass in the GC-treated rats is partially explained by their hypophagic behavior, which was evident on the third day of dexamethasone treatment in both male groups and in the 3-month-old female rats, and was obvious at the end of treatment in all of the groups. GC-induced hypophagy may be a result of anorexigenic insulin and leptin effects on the hypothalamus, as the concentration of both hormones is elevated in adult dexamethasone-treated rats (Rafacho et al. 2008a, 2011; Chimin et al. 2014). There is evidence for increased plasma insulin and leptin values (Caldefie-Chézet et al. 2001) in young (3 months old) and old (18, 24, and 26 months old) male rats. It has been reported that older female rats (21-weekold) lose body mass when treated with a dexamethasone infusion of 125 ␮g/kg b.m. for 7 continuous days, which was accompanied by no changes in food intake (Tomas 1998). In this respect, it is possible that the water balance may corroborate to the reduction of body mass. Dexamethasone (2 mg/kg b.m.) treatment markedly increases water and sodium excretion without affecting water intake, thereby producing negative water and sodium balances (Thunhorst et al. 2007). Thus, an excess of GCs appears to negatively impact body mass gain in an age- and gender-independent manner, which may be associated with the anorexigenic insulin effects. We did detect significant increases in fasting blood glucose concentration in both 3- and 12-month-old male and female rats subjected to the GC treatment. The augmented blood glucose levels were of higher magnitude in the older animals. These data indicated an additional degree of vulnerability for aged rats, independent of gender, to the GC effects on glucose homeostasis that is in accordance with previous studies showing a more pronounced effect of dexamethasone to cause increased fasting glycemia in male rats with 18 to 26 month old (Novelli et al. 1999; Caldefie-Chézet et al. 2001). Additionally, both groups of dexamethasone-treated male rats (MD3 and MD12) became glucose intolerant and less responsive to insulin action, based on the ipGTT and the AUC ipITT data, suggesting an effective GC-induced impairment of glucose homeostasis. The effects of GCs on the ␤-cells of adult male rats are well known, and although the islets from these rats are more responsive to glucose, this is insufficient to avoid the development of glucose intolerance (Giozzet et al. 2008; Rafacho et al. 2008a, 2010, 2011). Age per se does not appear to affect the basal glycemic values, but might have a slight effect on glucose tolerance in 12-month-old male rats, which is expected considering that aging naturally impairs the insulin response to glucose challenges (Reaven et al. 1983; Novelli et al. 1999). The effects of GCs on glucose tolerance and insulin sensitivity appear to be more apparent in older male animals. This could be attributed at least in part to the combination of reduced ␤-cell function (Reaven et al. 1983; Published by NRC Research Press

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Fig. 7. Serum lipids and hepatic triacylglycerol content. Serum triacylglycerol levels in 3- and 12-month-old (A) male and (D) female rats. The triacylglyceridemia was significantly increased all of the glucocorticoid (GC)-treated rats. Serum total cholesterol levels in 3- and 12-month-old (B) male and (E) female rats. No significant changes were observed among the groups. Hepatic triacylglycerol content in 3- and 12-month-old (C) male and (F) female rats. Note the elevation of fat content in the liver from MD12 and both female groups of GC-treated rats. The values are the mean ± SEM; n = 8 rats for A, B, D, F; and 6 fragments from 2 animals for C and F; *, p < 0.05 compared with the respective control group (effect of dexamethasone); #, p < 0.05 compared with the respective control group (effect of age). The data were analyzed using either one-way ANOVA followed by a Tukey post-hoc test, or Kruskal–Wallis non-parametric ANOVA followed by a Dunn post-hoc test.

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Fig. 8. Pancreatic ␤-cell mass. (A and C) Panoramic view of pancreas sections from all of the groups. (B and D) Absolute ␤-cell mass in 3- and 12-month-old male and female rats, respectively. An increase in the ␤-cell mass was observed in both MD3 and MD12 rats (significantly only for MD3 rats). The tissues were immunostained with insulin. The values are the mean ± SEM from 5 rats per group; *, p < 0.05 compared with the respective control group (effect of dexamethasone). The data were analyzed using either one-way ANOVA followed by a Tukey post-hoc test, or Kruskal–Wallis non-parametric ANOVA followed by a Dunn post-hoc test.

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Novelli et al. 1999) and increased ratio of plasma proinsulin to insulin hormone (Shimizu et al. 1996; Gama et al. 2000). Indeed, the ex vivo insulin secretion data obtained with isolated islets from the older male rats treated with dexamethasone (MD12), revealed lower GSIS as well as reduced capability to increment the insulin secretion, compared with the 3-month-old animals (MD3), indicating a lack of proper response to glucose when the body is challenged with GCs. These data are corroborated by evidence suggesting a decline in insulin secretion in older male rats treated with GCs (Reaven et al. 1983; Novelli et al. 1999). Thus, an excess of GCs affects glucose homeostasis in both 3- and 12-month-old male rats, with a milder effect observed in older rats. Adult female rats (FD3) were less prone to the side effects of dexamethasone on glucose homeostasis than the older rats. In fact, the 3-month-old female rats did not develop glucose intolerance. This can be explained, at least in part, by the increased capability of islets to respond to a high glucose concentration as well as by the absence of discernible peripheral insulin resistance. The augmented responsiveness to basal glucose concentration, as indicated by the insulin secretion data, suggests why these adult female rats were hyperinsulinemic. The time-course evaluation of the blood glucose decay during the first 8 min of the ipITT assay may also explain why these animals were hyperinsulinemic. Although the KITT data revealed no significant changes in the glucose decay constant during the linear phase (0 to 16 min), there still could be a mild degree of insulin insensitivity present in these animals. If this is correct, the compensatory hyperinsulinemia

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adaptively accounts for the increased metabolic insulin requirement, as observed in some insulin-resistance-related experimental models, such as obesity/metabolic syndrome (Fransson et al. 2014). The older, saline-treated female rats (FC12) exhibited reduced insulin sensitivity together with normoinsulinemia, normoglycemia, normotolerancia, and decreased absolute GSIS values compared with the FC3 rats. It is difficult to explain, but we suggest that the insulin secretion increment from basal to stimulatory glucose concentration (3.6-fold in the FC12 and 1.9-fold in the FD12) controls glucose homeostasis during glucose challenge. However, older female rats that received dexamethasone (FD12) had an augmented fasting glycemia and altered glucose tolerance, despite having the same degree of insulin insensitivity as the FC12 rats. This is consistent with the relatively reduced GSIS. The insulin secretion to high (16.7 mmol/L) glucose concentration in the FD12 rats was 58% of that in the FD3 rats, suggesting a deficient insulin secretory response that resulted in an insufficient insulin requirement to the increased insulin demand in these rats. Although these rats had higher insulinemia compared with the FC12 rats, we cannot exclude an increase of ratio of plasma proinsulin to insulin hormone as demonstrated previously by others (Shimizu et al. 1996; Gama et al. 2000) that could result in an inadequate control of glucose homeostasis. This age vulnerability to GCs resembles that of animals that are susceptible to instability in glucose homeostasis when exposed to GCs, such as in obesity (Ohneda et al. 1993; Holness et al. 2005; Fransson et al. 2014). Thus, the apparent protection observed in 3-month-old rats is not perPublished by NRC Research Press

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sistent in 12-month-old female rats, most likely as a consequence of different endocrine physiology. There are several reports indicating that 17␤-estradiol (E2) protects women and female rodents against acute fatty-acid-induced insulin resistance (Frias et al. 2001; Hevener et al. 2002). Activation of E2 receptor (ER␤) increases GSIS (Soriano et al. 2009) supporting a role for ER␤ as an insulinotropic receptor that may have important implications in physiological and pathological contexts. The menopausal period in rats occurs around 15 to 18 months of age (Sengupta 2011). Although we did not determine the plasma E2 concentrations in the 12-month old female rats, we cannot exclude the possibility that these rats had decreased plasma E2 levels and or ER␤ responsiveness that could account for their increased vulnerability to modulation of glucose homeostasis by GCs. Our findings with the pancreatic ␤-cell masses for the 3-monthold male rats (MD3) are in accordance with previous studies showing augmented ␤-cell masses in adult male rats subjected to dexamethasone treatment (Choi et al. 2006; Rafacho et al. 2010, 2011). The increase in ␤-cell mass is a known structural compensation to adapt to the reduction in insulin sensitivity and to provide the organism with the required insulin (Kahn et al. 2006; Weir and Bonner-Weir 2004). Age tended to elevate the absolute ␤-cell mass, which would be expected in instances of insulin resistance, but neither insulin sensitivity nor ␤-cell mass were significantly altered in the MC12 rats. Accordingly, the ␤-cell mass in the 12-month-old GC-treated rats was not significantly enhanced compared with the MC12 rats. Once again, the biological meaning of 58% increase cannot be ruled out, but despite this mild increase in ␤-cell mass, it was insufficient to prevent the manifestation of glucose intolerance in older male rats that had a high degree of insulin resistance after GC treatment. These data are in agreement with other studies demonstrating that a relative increase in ␤-cell mass does not necessarily prevent abnormalities in glucose homeostasis (Choi et al. 2006; Xue et al. 2007; Rafacho et al. 2010, 2011). The islet mass in females appeared to exhibit no changes except for a slight increase in the older rats. Considering that the 3-month-old rats (FC3 and FD3) and the saline-treated 12-monthold female rats had no significant alterations in their glucose tolerance, it is plausible that there is an absence of endocrine pancreas compensation at the structural level. The administration of dexamethasone to older female rats led to apparent hyperglycemia and glucose intolerance. This abnormality of glucose homeostasis in FD12 rats may be corroborated by the inability of the pancreas, which consists primarily of ␤-cells, to properly compensate for the GC-imposed insulin resistance by increasing the islet mass. This is in accordance with a study showing that pancreatic ␤-cells from 8-month old rats are more vulnerable to hyperglycemic environments than ␤-cells from young rats, as shown by enhanced ␤-cell apoptosis and diminished ␤-cell proliferation (Maedler et al. 2006). In summary, we demonstrated that 5 days of dexamethasone treatment induces glucose intolerance in both 3- and 12-monthold male rats and results in hyperinsulinemia, increased GSIS, and augmented ␤-cell mass. Three-month-old female rats are protected from the consequences of dexamethasone on glucose tolerance by increased insulin secretory capacity, but 12-month-old female rats reproduced all of the changes (glucose intolerance, dysglycemia, dyslipidemia, and insulin resistance) that were present in the 3- and 12-month-old male rats. Disclosure The authors declare that there are no conflicts of interest associated with this work.

Acknowledgements This study was supported by grants from the Brazilian foundations Fundação de Amparo a` Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e

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Tecnológico (CNPq) (471397/2011-3) and was also supported by the Instituto Nacional de Ciência e Tecnologia: Obesidade e Diabetes.

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Beta cell adaptation to dexamethasone-induced insulin resistance in rats involves increased glucose responsiveness but not glucose effectiveness. Pancreas, 22: 148–156. doi:10.1097/00006676-200103000-00007. PMID:11249069. Lin, K.H., Liou, T.L., Hsiao, L.C., and Hwu, C.M. 2011. Clinical and biochemical indicators of homeostasis model assessment-estimated insulin resistance in postmenopausal women. J. Chin. Med. Assoc. 74: 442–447. doi:10.1016/j.jcma. 2011.08.014. PMID:22036135. Published by NRC Research Press

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