Spironolactone, but Not Eplerenone, Impairs Glucose Tolerance in a Rat Model of Metabolic Syndrome

FULL PAPER Pharmacology

Spironolactone, but not Eplerenone, Impairs Glucose Tolerance in a Rat Model of Metabolic Syndrome

Tsuyoshi HOMMA1,2)*, Michio FUJISAWA2), Kiyoshi ARAI2), Marie ISHII2), Toshio SADA2) and Masahiro IKEDA1)

1)Department of Veterinary Pharmacology, Faculty of Agriculture, University of Miyazaki, 1–1 Gakuenkibanadai-nishi, Miyazaki-shi, Miyazaki, 889–2192, Japan 2)Biological Research Laboratories, R&D Division, Daiichi-Sankyo Co., Ltd., 1–2–58 Hiromachi, Shinagawa-ku, Tokyo 140–8710, Japan

(Received 9 January 2012/Accepted 1 April 2012/Published online in J-STAGE 13 April 2012)

Abstract. Although some clinical studies have suggested that spironolactone (SPL), a mineralocorticoid receptor (MR) antagonist, appears to increase the blood glucose levels, experimental studies have not supported this notion. Here, we investigated the effect of SPL on blood glucose levels in SHR/NDmcr-cp(cp/cp) (ND) rats, an animal model of metabolic syndrome, in comparison with that of eplerenone (EPL), another MR antagonist. At the same dose of 100 mg/kg, SPL and EPL increased the urinary sodium-to-potassium ratio to a comparable extent, indicating that both agents have similar renal MR antagonistic efficacy in ND rats. Interestingly, SPL but not EPL significantly increased the level of blood glucose. The oral glucose tolerance test revealed that treatment with SPL led to glucose intolerance. The levels of serum insulin and adiponectin, regulators of the blood glucose level, were virtually unaffected by treatment with SPL. On the other hand, SPL induced a marked increase in the blood level of aldosterone, known to be a risk factor for insulin resistance. These results demonstrate that in comparison with EPL, SPL characteristically impairs glucose tolerance in an animal model of metabolic syndrome, in association with a higher blood level of aldosterone. Key words: blood glucose, eplerenone, metabolic syndrome, mineralocorticoid receptor, spironolactone. doi: 10.1292/jvms.12-0007; J. Vet. Med. Sci. 74(8): 1015–1022, 2012

Aldosterone, a physiological ligand of the mineralo- sure, no clinical trials have focused on preventing further corticoid receptor (MR), is classically known to regulate metabolic deterioration and its complications. Spironolac- electrolyte homeostasis [12], and blood pressure. In addi- tone (SPL) and eplerenone (EPL), both of which are MR tion, several basic and clinical studies have indicated that antagonists currently used in a clinical setting, are widely aldosterone is directly involved in tissue damage in the recognized to be beneficial for patients with hypertension vasculature such as heart and kidney, through enhancement and heart failure [24, 25, 33]. However, the two drugs are of oxidative stress, inflammation, and fibrosis. known to have distinct pharmacological/pharmacokinetic Much attention has recently been focused on the relation profiles; EPL shows greater MR selectivity and has non- between aldosterone and impairment of glucose metabolism genomic properties, and SPL has active metabolites [28]. [7]. Glucose intolerance and diabetic mellitus are often Regarding the effect on glucose metabolism, EPL has been detected in patients with primary aldosteronism [11] due demonstrated to improve insulin sensitivity in obese model to adrenal adenoma or hyperplasia [1]. The impairment of mice [13, 14]. On the other hand, treatment with SPL has glucose metabolism is known to be involved in the pathobi- been reported to increase the blood glucose levels in pa- ology in the metabolic syndrome that comprises a cluster of tients with resistant hypertension, which is defined as blood several abnormalities, including obesity, insulin resistance, pressure that remains above the goal despite concurrent use dyslipidemia, and hypertension. Elevated plasma aldoste- of three antihypertensive agents of different classes [6]. rone levels are detected in metabolic syndrome [4], and Also, Yamaji et al. have shown that SPL increases HbA1c in moreover, experimental studies have produced a body of patients with mild chronic heart failure. These data indicate evidence for the involvement of aldosterone in the patho- that the results of experimental studies have not supported genesis of certain components of metabolic syndrome, such the clinical findings in terms of the effects of MR antago- as adipocyte dysfunction [5, 13] and insulin resistance [7]. nists on blood glucose level. Apart from the considerably beneficial effects of MR We considered that further investigations would be nec- blockade on metabolic syndrome through improvement of essary to compare the effect of SPL with that of EPL on glucose intolerance as well as suppression of blood pres- blood glucose levels in animal models. In the present study, therefore, we examined the effect of SPL on blood glucose levels in SHR/NDmcr-cp(cp/cp) (ND) rats, an animal model *Correspondence to: Homma, T., Biological Research Laborato- of metabolic syndrome (known to spontaneously develop ries, R&D Division, Daiichi-Sankyo Co., Ltd., 1–2–58 Hiroma- hypertension, obesity, and hyperlipidemia), in comparison chi, Shinagawa-ku, Tokyo 140–8710, Japan. with that of EPL, another MR antagonist. e-mail: [email protected] ©2012 The Japanese Society of Veterinary Science 1016 T. HOMMA ET AL.

MATERIALS AND METHODS Japan). The area under the curve (AUC) of blood glucose was calculated for each group. Experimental materials: SPL was purchased from Shang- Statistical analysis: Data are expressed as mean ± SE. hai FWD Chemicals Co., Ltd., (Shanghai, China). EPL was Comparisons between WKY-CONT and WKY-SPL100, extracted from Selara® tablets (purchased from Pfizer Japan and also between WKY-CONT and ND-CONT were per- Inc., Tokyo, Japan), and 0.5% (w/v) methylcellulose (MC) formed by t-test. Statistical comparison between ND-CONT solution was purchased from Wako Pure Chemical Indus- and the drug-treated ND groups, and WKY-CONT and the tries, Ltd. (Osaka, Japan). drug-treated ND groups, were performed by Dunnett’s test. Experimental protocol: All experiments were carried out Differences were considered to be statistically significant at in accordance with the Animal Experimentation Guidelines P<0.05. of Daiichi-Sankyo Co., Ltd. The rats were kept in a room at 55 ± 5% relative humidity and 23 ± 1°C under a 12 hr light/ RESULTS dark cycle, and allowed free access to diet (FR-2, Funabashi Farm Co., Ltd., Chiba, Japan) and water. Increase of uNa+/K+ ratio in ND-SPL100 is similar in Male SHR/NDmcr-cp(cp/cp) rats (ND, 15 weeks of age) extent to that in ND-EPL100: Once aldosterone binds to and WKY/Izm rats (WKY, 15 weeks of age) as non-obese MR on renal epithelial cells, urinary Na+ reabsorption and controls were purchased from Japan SLC Inc. (Shizuoka, Ja- K+ excretion occur, and therefore the uNa+/K+ ratio becomes pan). At 18 weeks of age, the rats were divided into 6 groups: lower [16, 27]. Table 1 summarizes the uNa+/K+ ratio as ND treated with MC solution (ND-CONT, n=6), ND treated well as body weight, urinary volume, food intake, and water with 30 mg/kg SPL (ND-SPL30, n=6), ND treated with 100 intake in the experimental groups on Day 15. In comparison mg/kg SPL (ND-SPL100, n=6), ND treated with 100 mg/ with the WKY-CONT group, the uNa+/K+ ratio sin the ND- kg EPL (ND-EPL100, n=6), WKY treated with MC solution CONT group was somewhat higher. Although the reason for (WKY-CONT, n=6) and WKY treated with 100 mg/kg SPL this is still unclear, sodium diuresis might occur in ND rats (WKY-SPL100). We selected these doses of SPL, because because of their hypertensive state. In comparison with the 30–100 mg/kg of SPL showed antihypertensive effect in ND ND-CONT group, a significantly higher uNa+/K+ ratio was rats in our preliminary study. SPL and EPL were suspended observed in both the ND-SPL100 and ND-EPL100 groups, in 0.5% (w/v) MC solution and administered at 2 ml/kg body the degree of elevation being comparable, suggesting that weight by gastric gavage once a day for 3 weeks from the both drugs have similar inhibitory potency on MR signaling grouping day. We defined the grouping day as Day 1. in renal epithelia. Measurement of urinary parameters and blood chemical SPL, but not EPL, increases the blood glucose level and parameters: The rats were placed individually in metabolic improves lipid profiles in ND rats: On Day 17, blood glucose cages, and 24-hr urine was collected on Day 15. Urinary and lipid parameters were measured under normal feeding sodium and potassium concentrations were measured using conditions, and the results have been summarized in Fig. the STAX-2 system (Techno Medica Co., Ltd., Kanagawa, 1. In comparison with WKY-CONT, the ND-CONT group Japan) and urinary sodium-to-potassium (uNa+/K+) ratios showed a significantly higher blood glucose level (Fig. 1A). were calculated. Water intake and food intake were also Although SPL had no effect on the blood glucose level in measured at the time of urine collection. WKY rats, administration of SPL to ND rats dose-depend- Blood was collected from the jugular vein on Day 17. ently and significantly increased the levels. In contrast, EPL Serum aldosterone (DPC Aldosterone RIA Kit, Diagnostic had no effect on the blood glucose level (Fig. 1A) in ND rats. Products Corporation, Los Angeles, CA, U.S.A.), corticos- The TG level in ND rats also increased, and this increase was terone (COAT-A-COUNT RAT CORTICOSTERONE, Di- significantly reduced by treatment with SPL, and tended to agnostic Products Corporation) were measured by radioim- be reduced in the ND-EPL100 group (Fig. 1B). In compari- munoassay. Insulin (REBIS Insulin Kit, Shibayagi, Gunma, son with WKY rats, the T-Cho level was significantly higher Japan) and adiponectin (Rat Adiponectin ELISA Kit, CirLex and HDL-C was lower in ND rats, respectively, although Co., Ltd., Nagano, Japan) were measured by ELISA. Blood the values were within the normal ranges (Fig. 1C and 1D). chemical parameters such as triglyceride (TG), total choles- Treatment of ND rats with SPL decreased T-Cho to the level terol (T-Cho), high density lipoprotein cholesterol (HDL-C) in WKY rats (Fig. 1C), but EPL had no significant effect and blood glucose, were measured using an automated bio- on T-Cho. Neither SPL nor EPL had a significant effect on chemical serum analyzer (JCA-BM2250, JOEL Ltd., Tokyo, HDL-C (Fig. 1D). These data indicate that SPL characteristi- Japan). cally increases blood glucose in ND rats. Oral glucose tolerance test: An oral glucose tolerance SPL, but not EPL, induces impairment of glucose toler- test (OGTT) was performed after 16 hr of fasting on Day ance in ND rats: Next, we performed the OGTT on Day 12. 12. Glucose solution at 2 g/kg (OTSUKA GLUCOSE IN- The fasting blood glucose levels (at 0 min after administra- JECTION 50%, Otsuka Pharmaceutical Co., Ltd., Tokyo, tion) were comparable among all experimental groups. The Japan) was administered by gastric gavage. Blood glucose OGTT showed that the blood glucose concentration increased levels were measured at 0, 30, 60, 120, 180, 240 and 360 quickly, peaked at around 60 min, and then returned to the min after glucose administration using a blood glucose meter basal level within 300 min after glucose administration in (GLUTEST®, Sanwa Kagaku Kenkyusho Co., Ltd., Nagoya, each experimental group, except for the ND-SPL100 group GLUCOSE INTOLERANCE BY SPIRONOLACTONE 1017

Table 1. body weight, urinary parameters, food and water intake on Day 15 WKY-CONT WKY-SPL100 ND-CONT ND-SPL30 ND-SPL100 ND-EPL100 (n=6) (n=6) (n=6) (n=6) (n=6) (n=6) Body weight (g) 418 ± 4 420 ± 3 550 ± 5 b) 529 ± 10 c) 509 ± 7 c) 548 ± 5 c) Urinary volume (g) 12.2 ± 1.5 18.0 ± 2.6 26.9 ± 2.0 b) 27.9 ± 3.9 c) 28.1 ± 5.6 c) 32.0 ± 4.0 c) uNa+/K+ ratio 0.55 ± 0.01 0.80 ± 0.08 a) 0.65 ± 0.03 b) 0.78 ± 0.08 c) 0.88 ± 0.08 c, d) 0.92 ± 0.03 c, d) Food intake (g/24 hr) 24.5 ± 1.3 20.3 ± 1.8 26.3 ± 2.6 25.2 ± 0.9 20.9 ± 1.0 c, d) 26.1 ± 0.7 Water intake (g/24 hr) 35.9 ± 0.8 41.2 ± 3.3 37.9 ± 2.9 40.6 ± 3.2 42.0 ± 5.4 43.6 ± 2.7 uNa+/K+ ratio: urinary sodium-to-potassium ratio. Data are expressed as mean ± SE. a): P<0.05 in comparison between WKY-CONT and WKY-SPL100 by t-test. b): P<0.05 in comparison between WKY-CONT and ND-CONT by t-test. c): P<0.05 vs. WKY-CONT by Dunnett’s test. d: P<0.05 vs. ND-CONT by Dunnett’s test.

Fig. 1. SPL improves lipid parameters, but increases blood glucose in ND rats. SPL, EPL or vehicle was administered orally to ND or WKY rats once a day during the experimental period. On Day 17, blood was collected from the jugular vein and blood glucose (A), triglyceride (B), total cholesterol (C) and HDL- cholesterol (D) were measured. The values are given as mean ± SE. n=6 for each group. **, P<0.01, *, P<0.05 compared with ND-CONT. ††, P<0.01, †, P<0.05 compared with WKY-CONT. NS, not significant. 1018 T. HOMMA ET AL.

Fig. 2. Administration of SPL, but not EPL, exacerbates glucose intolerance in ND rats. SPL, EPL or vehicle was administered orally to ND or WKY rats once a day during the experimental period. On Day 12, the rats were fasted for 16 hr and then glucose solution was administered at 2 g/kg by gastric gavage. Blood glucose in each group of ND rats (A) and WKY rats (B) was measured before dosing (0), and at 30, 60, 120, 180, 240, and 360 min after oral administration of glucose solution, and the AUC of blood glucose (C) was calculated. The values are given as mean ± SE. n=6 for each group. **, P<0.01, *, P<0.05 compared with ND-CONT. ††, P<0.01, †, P<0.05 compared with WKY-CONT. #, P<0.05 for comparison between WKY-CONT and WKY-SPL100.

(Fig. 2A and 2B). On the other hand, in the ND-SPL100 WKY rats. Administration of neither SPL nor EPL exerted group, the blood glucose level peaked at around 120 min and any significant effects on the serum insulin levels in ND rats then gradually returned toward the original level, although (Fig. 3A). the level was still higher than that at the baseline at 360 min The serum level of adiponectin, a reduction of which is after administration (Fig. 2A). The AUC value calculated known to cause insulin resistance [23], was also measured. from Fig. 2A and 2B, an index of glucose tolerance, was sig- The level in the ND-CONT group was higher than that in nificantly increased by SPL in both WKY and ND rats (Fig. the WKY-CONT group (Fig. 3B). Although a slight increase 2C). In contrast, EPL had no significant effect on the AUC was evident in the ND-SPL30 group, the adiponectin levels value in ND rats (Fig. 2C). These results indicate that ND in both the ND-SPL100 and ND-EPL100 groups did not dif- rats have impaired glucose tolerance, and that this impair- fer from that in the ND-CONT group (Fig. 3B). ment is dramatically exacerbated by administration of SPL. SPL increases the serum aldosterone concentration and SPL does not affect serum insulin or adiponectin concen- reduces the serum corticosterone concentration in ND rats: tration in ND rats: On Day 17, we measured serum insulin On Day 17, the serum aldosterone and corticosterone con- in the experimental groups. As shown in Fig. 3A, ND rats centrations were also measured. The level of aldosterone in showed marked hyperinsulinemia, compared with that of the ND-CONT group was significantly higher than that in GLUCOSE INTOLERANCE BY SPIRONOLACTONE 1019

Fig. 3. Effects of administration of SPL and EPL on serum insulin and adiponectin concentrations in ND rats. SPL, EPL or vehicle was administered orally to ND or WKY rats once a day during the experimental period. On Day 17, serum insulin (A) and adiponectin (B) levels were measured by ELISA. The values are given as mean ± SE. n=6 for each group. *, P<0.05 compared with ND-CONT. ††, P<0.01 compared with WKY-CONT. NS, not significant.

Fig. 4. Effects of administration of SPL and EPL on serum aldosterone and corticosterone concentrations in ND rats. SPL, EPL or vehicle was administered orally to ND or WKY rats once a day during the experimental period. On Day 17, serum aldosterone (A) and corticosterone (B) concentrations were measured by RIA. The values are given as mean ± SE. n=6 for each group. **, P<0.01, *, P<0.05 compared with ND-CONT. ††, P<0.01, †, P<0.05 compared with WKY-CONT. NS, not significant. the WKY-CONT group (Fig. 4A). The serum aldosterone tional glucocorticoid in rodents, in the experimental groups concentration in the ND-SPL100 group was significantly have been summarized in Fig. 4B. In comparison with the increased relative to that in the ND-CONT group (Fig. 4A). WKY-CONT group, the level of corticosterone in the ND- On the other hand, EPL had no effect on the serum aldoste- CONT group was significantly higher (Fig. 4B). In the ND- rone level even at the same dosage as that of SPL. SPL also SPL100 group, the level was significantly lower than that in increased the serum aldosterone level in WKY rats, but the the ND-CONT group (Fig. 4B). On the other hand, EPL did extent of the increase was smaller than that in ND rats (Fig. not significantly affect the serum corticosterone level (Fig. 4A). 4B). The serum concentrations of corticosterone, a major func- 1020 T. HOMMA ET AL.

DISCUSSION metabolites, whereas SPL is metabolized quickly, generating various active metabolites. Many pharmacological effects of ND rats are congenic animals harboring a nonsense mu- SPL are thought to be mediated by these metabolites. Higher tation introduced into the leptin receptor [29]. ND rats are concentrations of canrenone, a metabolite of SPL, were known to spontaneously develop symptoms of metabolic observed in liver, adrenals, kidney and testis in comparison syndrome, including obesity, hypertension, hyperlipidemia with that in plasma. Furthermore, the retention of radioactive and diabetes [3, 20], accompanied by age-related renal materials was observed in the adrenals after oral administra- injury [21]. In comparison with non-obese genetic control tion of 14C-labeled SPL [15], indicating that active metabo- animals, aldosterone excess has also been reported in ND lites were practically distributed in the adrenal glands [15]. rats [21]. These symptoms are known to be frequently ob- If these active metabolites affect MR in the periphery, aldo- served in patients with metabolic syndrome, and therefore sterone release may occur as a compensated action. Further we considered ND rats to be an appropriate animal model of studies are required to clarify the mechanisms underlying the metabolic syndrome. differential actions of MR antagonists on the blood level of In the present study, administration of SPL significantly aldosterone. elevated the levels of blood glucose with impairment of glu- The increased blood glucose levels resulting from treat- cose tolerance in ND rats, whereas EPL at a similar renally ment with SPL in ND rats in our present study contrasted effective dosage did not. On the other hand, serum insulin with results obtained from mice with high-energy diet- levels in rats treated with SPL were not significantly dif- induced obesity [31], in which SPL reduced blood glucose ferent from those in rats treated with EPL, suggesting that levels and improved glucose tolerance. However, the two SPL impaired insulin signaling. Previously, the CHARM models are quite different in that mice with high-energy diet- study has indicated that treatment of patients with SPL is induced obesity do not have an increased serum aldosterone associated with the development of diabetes mellitus [26]. level, whereas the serum aldosterone level in our present Furthermore, SPL has been reported to increase blood glu- model rats was four times higher than in control WKY cose levels in patients with type 2 diabetes mellitus [8]. Also, rats, reflecting results that have been obtained in humans. Yamaji et al. have shown that SPL increases the level of Moreover, methods used for oral administration of SPL dif- HbA1c in patients with chronic heart failure, whereas EPL fered between the two studies. In the mouse study, SPL was does not [32]. Taken together, these data suggest that SPL, administered by a subcutaneously implanted drug release but not EPL, increases the blood glucose level in association tablet, whereas in our study the drug was administered orally with glucose intolerance. by gastric gavage, thus resembling the clinical situation. There are some plausible reasons for the differences in MR blockade not only lowers blood pressure but also the effects of SPL and EPL on the blood glucose level. Since ameliorates cardiac morbidity and mortality in patients with MR activation by aldosterone plays a well-recognized role heart failure [24, 25, 33]. Furthermore, accumulated evidence in insulin resistance [7], increased aldosterone levels in re- indicates that MR antagonists reduce albuminuria in patients sponse to treatment with SPL would be a notable effect. If with chronic kidney disease or diabetic nephropathy [2, 18]. higher aldosterone levels resulted in insufficient blockade of These results suggest that treatment of metabolic syndrome MR, then insulin resistance would be expected to occur. Al- with MR antagonists would have various beneficial effects, ternatively, non-specific binding of SPL and its metabolites reducing the complications and risks of cardiovascular dis- to the sex steroid hormone [17] and glucocorticoid recep- ease. However, our present findings indicate that SPL has tors could exacerbate insulin resistance. EPL was originally the potential to increase the blood glucose level associated identified during the screening of SPL analogues for more with glucose intolerance in metabolic syndrome. Although selective binding to MR with minimal binding to other it remains unclear the influence of blood glucose elevation steroid receptors [9]. Therefore, in comparison with EPL, by SPL on clinical outcome, SPL has been reported to in- SPL shows 100- to 1,000-fold higher binding affinity for crease blood glucose levels in humans with type 2 diabetes glucocorticoid, androgen, and progesterone receptors [9]. and to impair endothelial function due to failure of glycemic Since glucocorticoid receptor activation is widely known to control [8]. In terms of hyperglycemia, EPL is considered increase blood glucose levels and it has also been reported to have a superior profile. However, administration of EPL that progesterone contributed to insulin resistance [17, 30], is contraindicated in diabetic patients with albuminuria, the lower selectivity of SPL to MR might be attributed to the because of the risk of hyperkalemia. Recently, some novel difference. The decrease in the serum corticosterone concen- non-steroidal MR antagonists with minimized unfavorable tration resulting from treatment with SPL, as observed in this effects have been reported [10, 19, 22]. Such a new class study, may reflect a feedback mechanism between the serum of MR antagonist may be a novel therapeutic option for pa- glucocorticoid level and activation of its receptor. tients with metabolic syndrome. In the present study, we found that SPL increased blood aldosterone levels, whereas EPL did not. Although the ACKNOWLEDGMENT. We would like to thank Dr. Toshiro reason for this is still unclear, a difference in the resulting Shibano in Daiichi Sankyo Co., Ltd., for his special support level of aldosterone might be explained by the view of the during our study. differences in the physicochemical and pharmacokinetic properties of SPL and EPL. EPL does not produce active GLUCOSE INTOLERANCE BY SPIRONOLACTONE 1021

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