Canadian Journal of Physiology and Pharmacology
Ameliorative effect of low-dose spironolactone on obesity and insulin resistance is through replenishment of estrogen in ovariectomized rats
Journal: Canadian Journal of Physiology and Pharmacology
Manuscript ID cjpp-2018-0416.R1
Manuscript Type: Article
Date Submitted by the 06-Oct-2018 Author:
Complete List of Authors: Olatunji, Lawrence; University of Ilorin, Ilorin, Nigeria, Cardiovascular Research Laboratory, Department of Physiology, ; Kyungpook National University DraftSchool of Medicine, Cardiovascular Research Laboratory Adeyanju, Oluwaseun; University of Ilorin, Ilorin, Nigeria, Cardiovascular Research Laboratory, Department of Physiology, Michael, Olugbenga; Bowen Univesity, Iwo, Physiology Usman, Taofeek; University of Ilorin College of Health Sciences Tostes, Rita; University of São Paulo, Ribeirao Preto Medical School Soladoye, Ayodele; University of Ilorin, Ilorin, Nigeria, Cardiovascular Research Laboratory, Department of Physiology,
Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue:
Cardiometabolic syndrome, Estrogen replacement therapy, GSK-3, Keyword: Mineralocorticoid receptor, Spironolactone
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1 Ameliorative effect of low-dose spironolactone on obesity and insulin 2 resistance is through replenishment of estrogen in ovariectomized rats
3 Lawrence A. Olatunji1, Oluwaseun A. Adeyanju1,2, Olugbenga S. Michael1,3, 4 Taofeek O. Usman1,4, Rita C. Tostes5, Ayodele O. Soladoye1.
5 1Cardiovascular Research Laboratory, Department of Physiology, College of Health Sciences, 6 University of Ilorin, Ilorin, Nigeria.
7 2Cardiometabolic Research Unit, Department of Physiology, Afe Babalola University, Ado- 8 Ekiti, Nigeria.
9 3Cardiometabolic Research Unit, Department of Physiology, College of Health Sciences, Bowen 10 University, Iwo, Nigeria.
11 4Cardiometabolic Research Unit, Department of Physiology, College of Health Sciences, Osun 12 State University, Osogbo, Nigeria.
13 5University of São Paulo, Ribeirao Preto Medical School, Ribeirao Preto, São Paulo, Brazil 14 Draft 15 Running title: Spironolactone replenishes estrogen and suppresses GSK-3 in OVX rats
16 *Address correspondence to: 17 Lawrence A. Olatunji, Ph.D. 18 Department of Physiology, 19 Faculty of Basic Medical Sciences, College of Health Sciences, 20 University of Ilorin,P.M.B. 1515, Ilorin, 240003, Nigeria. 21 Tel: +2348035755360 22 E-mail: [email protected] 23
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25 Disclosure Statement: All authors have filled the ICMJE Conflict of Interest Form and the 26 authors have no conflict of interest to declare.
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31 Abstract: Women have a lower incidence of cardiovascular diseases (CVD) than men at a similar
32 age but the reverse is the case after menopause, indicating a possible protective effect of estrogen
33 on cardiometabolic function. Although various hormonal therapies have been formulated to
34 combat the CVD risks in postmenopausal state, the beneficial effects have not been consistent.
35 Obesity with insulin resistance is closely linked to CVD risks while ovariectomized rodents have
36 been shown to mimic obese-insulin resistant (IR) state. We therefore hypothesized that low-dose
37 spironolactone, would ameliorate obese-IR in estrogen-deprived rats by replenishing estrogen
38 and suppressing elevated glycogen synthase kinase-3 (GSK-3). Ten weeks old female Wistar rats
39 were divided into four groups; sham-operated (SHM), spironolactone (SPL; 0.25 mg/kg) and
40 ovariectomized (OVX) rats treated with or without spironolactone daily for 8 weeks. Results
41 showed that estrogen deprivation throughDraft ovariectomy caused increased body weight gain and
42 visceral adiposity that are accompanied by increased HOMA-IR, HOMA-β, 1-hr postload
43 glucose, glucose intolerance, platelet/lymphocyte ratio, plasma insulin, atherogenic dyslipidemia,
44 uric acid, GSK-3, corticosterone, aldosterone and depressed 17β-estradiol. However, treatment
45 of OVX rats with spironolactone ameliorated all these effects. Taken together, the results
46 demonstrate that treatment with low-dose spironolactone improves obesity and IR, which
47 appears to involve replenishment of estrogen and suppression of GSK-3 along with circulating
48 mineralocorticoid and glucocorticoid. The findings imply a positive cardiometabolic effect of
49 low-dose spironolactone usage in estrogen-deprived conditions.
50 51 Key words: Aldosterone, Cardiometabolic syndrome, Estrogen replacement therapy, GSK-3,
52 Mineralocorticoid receptor, Spironolactone
53
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54 Introduction
55 Cardiovascular disease (CVD) has been the major cause of death for several decades and is
56 expected to remain so until 2030 (Mathers and Loncar 2006). It has been shown that women
57 have a lower incidence of CVD than men at a similar age, but the incidence increases after the
58 onset of menopause (Vitale et al. 2009). A bilateral ovariectomy (OVX) in women has also been
59 shown to be associated with increased mortality from CVD (Rivera et al. 2009), which may be
60 due to the fact that estrogen’s cardioprotective role is lost (Ren and Kelly 2009). More so, loss of
61 ovarian function is associated with increased adiposity, along with metabolic pathologies such as
62 insulin resistance (IR) and type 2 diabetes (Carr et al. 2004).
63 Obesity and IR are the major predisposingDraft factors to comorbidities, such as CVD, type 2
64 diabetes, non-alcoholic fatty liver disease, neurodegenerative diseases, and several types of
65 cancer. The prevalence of obesity is still increasing worldwide and now affects a large number of
66 individuals. Obesity with insulin resistant state is considered an important risk factor for CVD
67 due to its negative impact on glucose and lipid metabolism (Ginsberg 2000) as well as
68 accumulated visceral adiposity. Hence obesity through increased adipocytokines results in
69 inflammation (Karmazyn et al. 2008) and IR (Choi et al. 2012). Ovariectomy in rodent is
70 considered to resemble this obese-IR state (Ko et al. 2013) as it results in increase of fat mass
71 (Rogers et al. 2009) and chronic inflammation accompanied by IR (Stubbins et al. 2012),
72 implying a connection between metabolic complications observed in estrogen deficiency and
73 CVD. IR is also a critical metabolic link among hypertension, dyslipidemia, type 2 diabetes,
74 obesity and atherosclerotic CVD (DeFronzo 2009).
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75 Corticosteroids, glucocorticoids and mineralocorticoids, are steroid hormones produced
76 primarily in the adrenal cortex, which have numerous effects and can act on nearly all cells in the
77 body (Taves et al. 2011). The adrenal cortex can secrete glucocorticoids as cortisol (in humans)
78 or corticosterone (in rodents) and mineralocorticoids, primarily as aldosterone. Recent studies
79 have demonstrated the important role of glucocorticoids (Gulliford et al. 2006) and
80 mineralocorticoids (Pimenta et al. 2013) in the pathogenesis of IR and cardiometabolic risk
81 factors associated with increased mortality. Mineralocorticoid receptor (MR) activation results
82 in altered insulin signaling mechanisms in the heart, vasculature, liver and skeletal muscle that
83 collectively alter the cellular glucose metabolism (Giacchetti et al. 2007). Furthermore, other
84 studies also reported that MR blockade improved IR, glucose tolerance, and decreased fasting
85 levels of triglycerides in obese rats Draft (Hirata et al. 2009). Spironolactone is a well-known
86 antagonist of MR and we have recently reported that a low-dose spironolactone has ameliorative
87 effects on the development of cardiometabolic disorders during gestational androgen excess
88 (Olatunji et al. 2017).
89 GSK-3 is a broadly expressed, well-conserved serine/threonine kinase, which can serve as a
90 negative modulator of insulin action on glycogen synthesis and, potentially, on glucose utility
91 (Eldar-Finkelman and Krebs 1997). Hence, GSK-3 has been implicated in the development of
92 IR, pancreatic β-cell dysfunction, diabetes and inflammation (Liu et al. 2010). It has also been
93 reported that glucocorticoids can also cause impaired insulin action through GSK-3-dependent
94 pathway (Ruzzin et al. 2005). Furthermore, estrogen actions might be mediated through GSK-3-
95 dependent pathway (Grisouard et al. 2007). We have also reported the involvement of elevated
96 GSK-3 levels in the pathogenesis of cardiometabolic disorder (Michael and Olatunji 2017).
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97 A fundamental issue faced by most postmenopausal women is the potential impact of estrogen
98 replacement therapy on the prevalence of CVD as different strategies have produced conflicting
99 outcomes (Manson et al. 2003). Usage of spironolactone to suppress hyperandrogenism in
100 premenopausal women with polycystic ovarian syndrome has been attributed to increased
101 aromatization of testosterone to estrogen (Haynes and Mookadam 2009). However, there is
102 paucity of information on the effect of spironolactone on obesity-IR induced by estrogen
103 deprivation and possible roles of estrogen/GSK-3 pathway. We therefore tested the hypothesis
104 that low-dose spironolactone would ameliorate OVX-induced obesity-IR by replenishing
105 estrogen and suppressing mineralocorticoid/glucocorticoid/ GSK-3 pathway.
106 Materials and methods
107 Animals. The study was conducted in accordanceDraft with the National Institute of Health Guide for
108 the Care and Use of Laboratory Animals, approved by the Institutional Review Board of the
109 University of Ilorin, and every effort was made to minimize both the number of animals used and
110 their suffering. Female Wistar rats (10 weeks old) were obtained from the animal house of the
111 College of Health Sciences, University of Ilorin (Ilorin, Nigeria) and kept under standard
112 environmental conditions of temperature, relative humidity, and dark/light cycle with
113 unrestricted access to standard rat chow and tap water.
114 Surgical procedure. After 1 week of acclimatization, animals were anesthetized (ketamine, 50
115 mg/kg i.p.) under aseptic conditions and underwent ovariectomy (OVX group) or sham surgery
116 (SHM group). The ovariectomy was performed by a ventral abdominal midline incision to access
117 the abdomen. Ovaries were bilaterally clamped and removed. The uterine horns were clamped
118 and the uterus was left intact. Then, the abdominal wall was sutured. After surgery, rats were
119 maintained under good conditions to recover. In the sham procedure, animals were anesthetized
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120 and the abdominal wall was opened like the OVX rats, the ovaries were exteriorized to create
121 similar stress, but they were not removed (Adeyanju et al. 2018). After a 1-wk resting period,
122 each group (SHM and OVX) was randomized into four new groups (n=5/group) based on the
123 treatment: sham-operated control (SHM), spironolactone (SPL), ovariectomized rats (OVX) and
124 OVX treated with SPL (OVX+SPL). Food and water intakes were monitored daily while body
125 weight was weekly.
126 Treatment. SHM and OVX groups received distilled water (vehicle; po) while the SPL- and
127 OVX+SPL-treated group received (po) 0.25 mg/kg bw spironolactone (Pfizer Inc., New York,
128 NY) daily. The treatment lasted for 8 weeks.
129 Sample preparation. At the end of treatment,Draft the rats were anesthetized with pentobarbital
130 sodium. Blood was collected by cardiac puncture into heparinised bottle and was centrifuged at
131 3000 rpm for 5min. Plasma was stored frozen until needed for biochemical assay.
132 Oral glucose tolerance test (OGTT), insulin resistance (IR), pancreatic β-cell function and
133 visceral adiposity. Glucose challenge test was performed 24 h before the end of the experiment.
134 The rats had 12 h overnight fast. Glucose (2 g/kg bw) was given (po). Blood sample was
135 obtained from the tail vein before glucose load and then sequentially after 30, 60, 90 and 120
136 min using a glucometer (ONETOUCH®-Life Scan, Inc., Milpitas, CA, USA). Glucose tolerance
137 was expressed as a function of the area under the curve (AUC) for oral glucose tolerance test
138 (OGTT) as previously described (Olatunji et al. 2012). The IR was determined using the
139 homeostasis model assessment for insulin resistance (HOMA-IR = fasting glucose (mmol/l) *
140 fasting insulin (μIU/l)/22.5) where as HOMA-β (20* fasting insulin (μIU/l) / fasting glucose
141 minus 3.5) while 1-h postload glucose level were used to determine pancreatic β-cell function
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142 (Bianchi et al. 2013). Triglyceride-glucose index was also used to determine IR (TyG-index = Ln
143 [fasting plasma triglycerides (mg/dl) × fasting plasma glucose (mg/dl)/2] (Simental-Mendia et al.
144 2008).
145 Visceral adiposity. Following blood samples collection, all intra-abdominal fat depots including
146 mesenteric, urogenital and retroperitoneal were dissected out and weighed immediately with an
147 electronic weighing scale after dissection to avoid evaporative weight loss.
148 Biochemical assays. Plasma corticosterone, aldosterone, 17β-estradiol and GSK-3 were
149 determined using ELISA kits obtained from Elabscience Biotechnology Co., Ltd. (Wuhan,
150 China) while insulin was determined using ELISA kit from RayBiotech Inc. (Georgia, USA). 151 Total cholesterol (TC) and triglycerideDraft (TG) were measured by standardized enzymatic 152 colorimetric methods using assay kit obtained from Fortress Diagnostics Ltd. Antrim, UK. High-
153 density lipoprotein cholesterol (HDL-C) was measured by enzymatic clearance assay (Daiichi
154 Pure Chemicals Co., Ltd., Tokyo, Japan) whereas low-density lipoprotein cholesterol (LDL-C)
155 was estimated using Ananandaraja formula. TC/HDL-C and TG/HDLC ratios were estimated as
156 indices of atherogenic dyslipidemia.
157 Statistical analysis. All data were expressed as means ± standard error of mean (SEM).
158 Statistical group analysis was performed with SPSS statistical software. One-way analysis of
159 variance (ANOVA) was used to compare the mean values of variables among the groups.
160 Bonferroni’s test was used to identify the significance of pairwise comparison of mean values
161 among the groups. Statistically significant differences were accepted at p < 0.05.
162
163
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164 Results
165 Effects of spironolactone (SPL) on body weight, visceral adiposity, food intake and water intake 166 in OVX rats.
167 After ovariectomy and 8 weeks of experimentation, OVX rats gained more weight than SHM
168 rats (Table 1 and Figure 1A), and they also had increased visceral fat deposition (Figure 1B)
169 suggesting that ovariectomy-induced estrogen deprivation leads to obesity in rats (Table 1 and
170 Figure 1B) SPL-treated rats had increased food intake but no significant change in body weight
171 and visceral adiposity compared to SHM rats (table 1 and fig 1). SPL treatment in OVX rats
172 attenuated both increases in body weight and visceral adiposity (table 1 and fig 1B). OVX rats
173 had significantly decreased water intake compared with SHM rats whereas SPL treatment in 174 OVX rats led to increased water intake comparedDraft with OVX rats (table 1).
175 Spironolactone improves postload glycemia in OVX rats.
176 OVX rats had elevated 1-hr postload glycemia whereas in SPL- and OVX+SPL-treated rats, 1-hr
177 postload glycemia was not affected when compared with the SHM rats. However, SPL treatment
178 in OVX rats attenuated the elevated 1-hr postload glycemia when compared with OVX rats.
179 There was no difference in 2-hr postload glycemia (fig 2A). The area under glucose tolerance
180 curve was higher in the OVX rats whereas in SPL- and OVX+SPL-treated rats, area under
181 glucose tolerance curve was not affected when compared with SHM rats (fig 2B). However,
182 treatment with SPL in OVX rats ameliorated the high area under glucose tolerance curve when
183 compared with the OVX rats (fig 2B).
184
185
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186 Spironolactone improves fasting insulinemia, IR, pancreatic β-cell function and TyG in OVX 187 rats.
188 The fasting glycemia was not significantly different among the groups (table 2). On the other
189 hand, there was significantly elevated fasting insulinemia, HOMA-IR, HOMA-β and TyG
190 indices in OVX rats compared with SHM rats (figs 3A-D), whereas fasting insulinemia, HOMA-
191 IR, HOMA-β and TyG indices were unchanged in the SPL group when compared with SHM rats
192 (figs 3A-D). However, the elevated fasting insulinemia, HOMA-IR, HOMA-β and TyG indices
193 observed in the OVX rats were attenuated by OVX+SPL treatment (figs 3A-D).
194 Spironolactone attenuates atherogenic dyslipidemia in OVX rats.
195 Spironolactone treatment led to reduction in triglyceride (TG) level whereas total cholesterol
196 (TC) and low density lipoprotein cholesterolDraft (LDL-C) levels were not affected compared with
197 SHM rats (table 2). However, TG, TC and LDL-C levels were elevated in OVX rats compared
198 with SHM rats (table 2). In addition, TG, TC and LDL-C levels were significantly attenuated in
199 the OVX+SPL-treated rats compared with the OVX rats (table 2). High-density lipoprotein
200 cholesterol (HDL-C) was significantly lowered in all the experimental groups compared with
201 SHM rats (table 2). Atherogenic lipid indices (TG/HDL-C & TC/HDL-C) were not altered in
202 SPL-treated rats compared with SHM rats whereas TG/HDL-C & TC/HDL-C ratio were elevated
203 in OVX rats compared with SHM rats (fig 4A&B). However, the atherogenic dyslipidemia
204 observed in OVX rats was attenuated by OVX+SPL treatment (figs 4A&B).
205 Spironolactone decreases pro-inflammatory markers in OVX rats.
206 Uric acid level was not affected among the experimental groups compared with SHM rats (fig
207 5A). Spironolactone treatment did not affect the platelet/lymphocyte ratio compared with SHM
208 rats whereas there was increased platelet/lymphocyte ratio in OVX rats compared with the SHM
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209 rats. However, the platelet/lymphocyte ratio was significantly attenuated in the OVX+SPL-
210 treated group compared with OVX rats (figs 5A&B).
211 Spironolactone attenuates GSK-3, circulating glucocorticoid, mineralocorticoid and replenishes 212 17β-estradiol in OVX rats.
213 Spironolactone treatment did not affect GSK-3 level in all experimental groups but led to
214 elevated aldosterone and corticosterone levels compared with SHM rats. Circulating
215 corticosterone, aldosterone and GSK-3 were elevated in OVX rats compared with SHM rats.
216 However, circulating corticosterone, aldosterone and GSK-3 were attenuated in OVX+SPL-
217 treated rats compared with OVX rats. Plasma 17β-estradiol level was not affected by SPL
218 treatment whereas OVX rats had reduced plasma 17β-estradiol levels when compared with SHM
219 rats. However, treatment with SPL in OVXDraft rats replenished 17β-estradiol (figs 6; 7A-C).
220
221 Discussion
222 The results from the present study provides evidence that the obesity and insulin resistance
223 observed in OVX rats is primarily, a consequence of estrogen deprivation, which are
224 accompanied by hyperinsulinemia, elevated atherogenic dyslipidemia, GSK-3, aldosterone and
225 corticosterone. The study also provides important findings that treatment with low dose
226 spironolactone, a mineralocorticoid receptor (MR) blocker, ameliorates the increased body
227 weight gain, visceral fat accumulation and insulin resistance, which are accompanied not only by
228 suppression of elevated mineralocorticoid (aldosterone), glucocorticoid (corticosterone) and
229 GSK-3 but also by replenishment of estrogen in estrogen-deprived rats. These findings imply
230 that mineralocorticoid receptor antagonists, such as spironolactone, would protect against obesity
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231 and insulin resistance in estrogen-deprived states and represents an adjuvant therapy in the
232 management of cardiometabolic disorders.
233 Spironolactone a non-selective MR antagonist has been in wide clinical use for several decades
234 and is moderately (~20-folds) more potent than a newer and selective MR antagonist, eplerenone
235 in binding to MR. Spironolactone also interacts with androgen and progestogen receptors
236 (Epstein and Calhoun 2011). Spironolactone is widely recognized to be beneficial in patients
237 with primary aldosteronism-related hypertension and heart failure, and the beneficial effects in
238 these patients are significantly greater than that of eplerenone demonstrating the role of non-MR
239 involvement in spironolactone’s beneficial effects (Epstein and Calhoun 2011). On the other 240 hand, treatment with spironolactone at Drafthigh dose has been reported to impair glucose regulation 241 in humans and in a rat model of metabolic syndrome (Homma et al. 2012). However, the finding
242 in this study that spironolactone improves glucose regulation/insulin resistance is in consonance
243 with other animal studies that used spironolactone at low dose (Olatunji et al. 2017). The
244 disparity in the effects of spironolactone on glucose metabolism is likely to be dose dependent
245 because previous studies have shown that non-MR binding of spironolactone requires a high
246 dosage (de Gasparo et al. 1987). The fact that, in this study, we employed a very low dose
247 spironolactone (0.25 mg/kg) has ruled out the likelihood of spironolactone interaction with other
248 nuclear receptors. Hence, the effects of spironolactone on glucose metabolism observed in this
249 study may at least be as a result of MR activation.
250 Epidemiological, clinical and molecular studies have shown that estrogen plays an important role
251 on metabolic homeostasis (Barros and Gustafsson 2011), and that estrogen-deprived state in both
252 menopausal women and rodents (Rogers et al. 2009) may have profound effects on glucose
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253 homeostasis and development of obesity which induces several subsequent clinical diseases such
254 as diabetes mellitus and CVD. This present study lends credence to the cardiometabolic
255 protective effects of estrogen. Spironolactone has also been shown to increase circulating
256 estrogen in women with polycystic ovarian syndrome-related glucose dysmetabolism (Dăneasă
257 et al. 2014). The increase in estrogen following spironolactone treatment in women with PCOS
258 has been attributed at least to increased peripheral aromatization of androgen to estrogen and
259 increased bioavailability and activity of estrogen (Haynes and Mookdam 2009). Hence, this
260 corroborates our observation in the recent study that treatment with spironolactone increases
261 circulating estradiol. Taken together, the ameliorative effect of spironolactone on
262 cardiometabolic alterations in OVX rats might be attributed to both blockade of
263 mineralocorticoid and replenishment of Draftestrogen.
264 The observation that estrogen deprivation in OVX rats induced increased body weight gain
265 which was accompanied by increased visceral fat deposition is consistent with other studies
266 (Sivasinprasasn et al. 2015) and also the finding that food intake in OVX rats was not altered
267 despite increased body weight gain is also consistent with other studies (Sivasinprasasn et al.
268 2015) but not in agreement with a study that reported decreased food intake in OVX rats
269 (Santollo et al. 2013). The observed decreased water intake in OVX rats is line with the report of
270 Santollo and colleagues (Santollo et al. 2013). This observation implies that the gain in body
271 weight and fat accumulation is unlikely due to central feeding control (table 1 and fig 1B). The
272 weight gain and increased visceral fat accumulation might be due to estrogen deprivation as it
273 has been indicated in previous studies in OVX rats (Sivasinprasasn et al. 2015). It seems
274 estrogen signaling contributes to the modulation and distribution of body fat mass probably
275 through its lipolytic effect rather than influencing the central mechanism of food intake. The
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276 observation that SPL treatment in OVX rats attenuated the body weight gain and visceral fat
277 accumulation agrees with recent studies in high fat diet-induced obesity using spironolactone at
278 20 mg/Kg/day (Armani et al. 2014). However, a study in a rodent model of type 2 diabetes
279 reported that spironolactone at a very high dose (50 mg/Kg/day) did not affect body weight gain
280 (Silva et al. 2015), providing other evidence that spironolactone may have divergent dose-
281 dependent pharmacological effects when administered at different doses.
282 The present study showed that OVX in rats induced disturbed metabolism that is associated with
283 disrupted glucose homeostasis regardless of normoglycemia in all the experimental groups.
284 Plasma levels of insulin were elevated in OVX rats in this study consistent with reports in OVX 285 rodents by previous investigators (SivasinprasasnDraft et al. 2015). Spironolactone ameliorated the 286 insulin level as well as improved insulin resistance as determined by HOMA-IR, TyG and
287 TG/HDL-cholesterol indices which is also consistent with previous studies (Olatunji et al. 2017).
288 Several factors such as elevated circulating aldosterone and corticosterone are involved in the
289 pathogenesis of insulin resistance and cardiometabolic disorder through the activation of MR.
290 Attenuation of the corticosteroids; aldosterone and corticosterone in this study by spironolactone,
291 suggest that IR and compensatory hyperinsulinemia in the OVX rats are due to activation of MR
292 receptor through both aldosterone/MR-and glucocorticoid/MR- dependent pathways. This
293 finding also indicates that MR blockade may represent effective pharmacological intervention in
294 the treatment of cardiometabolic disturbances in estrogen-deprived states.
295 The protection of the OVX+SPL rats from IR may be explained by the increase in E2 level
296 (Figure 7) and the blockade of the MR. The mechanism behind this might be the ability of
297 spironolacone to increase blood levels of estradiol by increasing peripheral conversion of other
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298 sex hormones to estradiol, a process referred to aromatization (Satoh et al. 2002). In this context,
299 spironolacone use might be a suitable adjuvant in estrogen-deprived states, especially in the
300 treatment of IR and hence, cardiometabolic disorder. This is because the dose of spironolactone
301 used in the present study led to replenishment of the E2 level in OVX rats which was
302 accompanied by improvements in obesity and IR.
303 The finding that IR induced by OVX is accompanied by increased atherogenic dyslipidemia
304 characterised by elevations of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-
305 C), triglyceride (TG) concentrations, decrease in high-density lipoprotein cholesterol (HDL-C)
306 and TG/HDL-C ratio which is a useful marker in identifying individuals at risk for developing 307 atherosclerotic CVD is noteworthy (MussoDraft et al. 2011).These disturbances in OVX rats are 308 consistent with previous studies in animals and humans (Mooradian 2009). The observed
309 derangements in plasma lipids may be a cause of depleted E2 levels in OVX animals.
310 It is interesting to note that OVX rats had increased platelet-lymphocyte ratio while there was no
311 significant difference in the plasma uric acid among the groups. Platelet-lymphocyte ratio has
312 been shown to be an inflammatory marker and this ratio was recently assessed in relation to
313 coronary artery disease (Temiz et al. 2014). Evidences exists that increased pro-inflammatory
314 markers (Tzoulaki et al. 2007) play considerable pathogenic role in the development of IR.
315 Increased pro-inflammatory activities have been observed in estrogen- deprived states (Lee et al.
316 2009). Treatment with spironolactone in OVX rats ameliorated the platelet-lymphocyte ratio, an
317 inflammatory response, which indicates that the inflammatory response in estrogen-deprived
318 state is mediated by MR activation.
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319 Glucocorticoids (GCs) are stress and catabolic hormones produced and released from the zona
320 fasciculata of the adrenal cortex. They are primarily responsible for mobilizing glucose to the
321 circulation from the liver and also by inhibiting the uptake and utilization of glucose in the
322 skeletal muscle and adipose tissue. Hence, IR and other CVD risk factors observed are concerns
323 in conditions with elevated circulating GCs or during GC-based therapy (Gulliford et al. 2006).
324 Estrogen deprivation in menopausal and premenopausal women has been shown to result in
325 increased circulating corticosteroids through activation of renin-angiotensin system (O'Donnell
326 et al. 2014). Corticosteroid/GSK-3 pathway has also been implicated in the development of IR
327 and associated kidney injury (Lee et al. 2009). Furthermore, estrogen action has been shown to 328 be modulated via GSK-3 dependent pathwayDraft (Liu et al. 2010). Obesity has also been linked to 329 elevated GSK-3 (Grisouard et al. 2007). Taken together, the fact that estrogen deprivation in
330 OVX rats resulted in obesity, increased body weight gain, inflammation, visceral fat
331 accumulation, atherogenic dyslipidemia that are accompanied by elevated corticosteroids and
332 GSK-3 suggests that estrogen deprivation-induced obesity and IR might be due to corticosteroid-
333 GSK-3 mediated pathway. Most importantly, treatment with spironolactone at 0.25 mg/kg
334 replenished estrogen in OVX rats and ameliorated increased body weight gain, visceral adiposity
335 and IR which are accompanied by suppressed corticosteroids and GSK-3. The replenishment of
336 estrogen in OVX rats by spironolactone treatment improves obesity/IR via suppression of
337 corticosteroid/MR/GSK-3 pathway and corticosteroid/GSK-3 pathway (fig 8). Suppression of
338 corticosteroids and MR blockade by spironolactone might decrease GSK-3 mediation, reducing
339 the activity of the kinase to disrupt glucose homeostasis or induce cardiometabolic disorders in
340 insulin resistant state.
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341 In conclusion, the present results demonstrate that treatment with low dose spironolactone
342 protects against obesity and insulin resistance by the replenishment of estrogen and suppression
343 of GSK-3 along with corticosteroids in estrogen deprived rats. These imply the prominent role of
344 MR and/or GSK-3 in the development of cardiometabolic disorder induced by estrogen
345 deprivation. Therefore, spironolactone may be an adjuvant pharmacological therapy for
346 cardiometabolic disorder induced by estrogen deprivation.
347
348
349
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486 Table 1: Effect of spironolactone treatment on body weight, food and water intake in 487 estrogen-deprived rats SHM SPL OVX OVX+SPL Final body weight (g) 217.2 ± 2.6 221.8 ± 8.5 241.3 ± 3.4* 228.1 ± 2.9# Food intake (g/kg bw) 65.2 ± 3.0 85.3 ± 5.7* 65.5 ± 5.9 58.0 ± 2.4 Water intake (ml/kg bw) 103.4 ± 1.6 113.9 ± 5.7 84.5 ± 7.4* 104.9 ± 2.1# 488 Data are expressed as mean ± SEM of 5 rats per group. Data were analyzed by one-way ANOVA followed by 489 Bonferroni post hoc test (*p<0.05 vs SHM; #p<0.05 vs OVX). 490
491 492 493 494 495 496 497 498 499 500 501
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502 Table 2: Fasting blood glucose and lipid profile SHM SPL OVX OVX+SPL FBG (mmol/l) 3.4 ± 0.5 3.7 ± 0.2 3.6 ± 0.2 3.4 ± 0.4 Triglyceride (mg/dl) 107.2 ± 7.2 80.8 ± 3.5* 156.7 ± 6.2* 117.6 ± 3.7# Total cholesterol (mg/dl) 62.9 ± 5.2 63.5 ± 4.3 100.1 ± 4.3* 82.6 ± 4.3*# HDL-cholesterol (mg/dl) 70.6 ± 9.4 43.8 ± 6.6* 23.2 ± 1.7* 19.8 ± 0.8* LDL- cholesterol(mg/dl) 40.5 ± 2.9 44.6 ± 1.5 63.8 ± 2.1* 46.5 ± 3.6# 503 Data are expressed as mean ± SEM of 5 rats per group. Data were analyzed by one-way ANOVA followed by 504 Bonferroni post hoc test (*p<0.05 vs SHM; #p<0.05 vs OVX). 505
506 FIGURE LEGENDS
507 Figure 1.Effect of spironolactone treatment on body weight (A) and visceral adiposity (B). SPL 508 treatment did not affect body weight and visceral adiposity compared with SHM rats. However, 509 there was significant increase in body weight and visceral adiposity in OVX rats compared with 510 SHM rats whereas, SPL treatment attenuated the body weight and visceral adiposity in OVX rats 511 compared with OVX rats. Data were analyzedDraft by one-way ANOVA followed by Bonferroni post 512 hoc test. Values are expressed as mean ± SEM of 5 rats per group (*p<0.05 vs SHM; #p<0.05 vs 513 OVX).
514 Figure 2.Effect of spironolactone treatment on oral glucose tolerance test (OGTT; A) and area 515 under curve (AUC) of OGTT (B).SPL treatment did not affect 1-hr postload glucose and AUC 516 compared with SHM rats. OVX rats had increased 1-hr postload glucose and AUC respectively 517 compared with SHM rats. The increase in 1-hr postload glucose and AUC was attenuated in 518 OVX+SPL– treated compared to OVX rats. Data were analyzed by one-way ANOVA followed 519 by Bonferroni post hoc test. Values are expressed as mean ± SEM of 5 rats per group (*p<0.05 520 vs SHM; #p<0.05 vs OVX).
521 Figure 3.Effect of spironolactone treatment on fasting insulinemia (A) HOMA-β (B) insulin 522 resistance (IR; C) and triglyceride index (TyG; D). Fasting insulinemia, HOMA-IR, HOMA-β 523 and TyG indices were unchanged in the SPL group when compared with SHM rats whereas there 524 was significantly elevated fasting insulinemia, HOMA-IR, HOMA-β and TyG indices in OVX 525 rats compared with SHM rats. However, the elevated fasting insulinemia, HOMA-IR, HOMA-β 526 and TyG indices observed in the OVX rats were attenuated by OVX+SPL treatment. Data were 527 analyzed by one-way ANOVA followed by Bonferroni post hoc test. Values are expressed as 528 mean ± SEM of 5 rats per group (*p<0.05 vs SHM; #p<0.05 vs OVX).
529 Figure 4. Effect of spironolactone treatment on atherogenic indices (A&B). SPL treatment did 530 not affect the atherogenic indices (TG/HDL-C & TC/HDL-C) compared with SHM rats. OVX 531 led to an increase in atherogenic dyslipidemia which was attenuated in the OVX+SPL–treated 532 rats. Data were analyzed by one-way ANOVA followed by Bonferroni post hoc test. Values are 533 expressed as mean ± SEM of 5 rats per group (*p<0.05 vs SHM; #p<0.05 vs OVX).
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534 Figure 5. Effect of spironolactone treatment on circulating pro-inflammatory markers 535 (A&B).SPL treatment did not affect PLT/LYM ratio compared with SHM rats whereas OVX 536 caused an increase in PLT/LYM ratio when compared with the SHM. OVX+SPL treatment 537 attenuated the increased PLT/LYM ratio observed in the OVX animals. Uric acid level was 538 comparable among the experimental groups compared with SHM rats. Data were analyzed by 539 one-way ANOVA followed by Bonferroni post hoc test. Values are expressed as mean ± SEM of 540 5 rats per group (*p<0.05 vs SHM; #p<0.05 vs OVX).
541 Figure 6.Effect of spironolactone treatment on circulating glycogen synthase kinase-3 (GSK- 542 3).SPL treatment did not alter circulating GSK-3 compared with SHM rats whereas OVX led to 543 increase in circulating GSK-3 compared with the SHM rats which was attenuated in the 544 OVX+SPL rats. Data were analyzed by one-way ANOVA followed by Bonferroni post hoc test. 545 Values are expressed as mean ± SEM of 5 rats per group (*p<0.05 vs SHM; #p<0.05 vs OVX).
546 Figure7. Effect of spironolactone treatment on circulating glucocorticoids (A), mineralocorticoid 547 (B) and 17β-estradiol (C). SPL treatment did not alter circulating 17β-estradiol but led to 548 increases in circulating glucocorticoid and mineralocorticoid compared with SHM rats. 549 However, OVX led to increases in circulating glucocorticoid and mineralocorticoid compared 550 with the SHM which were attenuated in the OVX+SPL rats. Data were analyzed by one-way 551 ANOVA followed by Bonferroni post hoc test. Values are expressed as mean ± SEM of 5 rats 552 per group (*p<0.05 vs SHM; #p<0.05 vsDraft OVX). 553 Figure 8. Schematic diagram of the possible mechanism by which SPL prevents obese-insulin 554 resistance and GSK-3. SPL; spironolactone, GSK-3; glycogen synthase kinase-3.
555
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SHM a SHM+SPL b OVX OVX+SPL * 27 260 * )
# bw 240 * * # 18 # 220 * Draft 200
Body weight (g) Body 9
180 Visceral Adiposity (g/kg Adiposity Visceral
160 0 0 1 2 3 4 5 6 7 8 SHM SHM+SPL OVX OVX+SPL Weeks
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SHM 8 SHM+SPL 800 OVX ) * OVX+SPL -1 6 600 # # 4 * 400 #
2 Draft200 AUC (mmol.min.l of OGTT
Postload glycemic changes (mmol/l) changes glycemic Postload 0 0 0 30 60 90 120 SHM SHM+SPL OVX OVX+SPL Time (min)
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#
#
25 β 5 HOMA- (%) (%) HOMA- Insulin (µU/ml) Insulin
0 Draft0 SHM SHM+SPL OVX OVX+SPL SHM SHM+SPL OVX OVX+SPL c d
9 * 6.2 * 6.0
6 5.8 # 5.6 TyG
HOMA- IR # 3 5.4
5.2
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a b 9 * 6 *
6 *# 4 *# Draft TC/HDL-C TG/HDL-C 3 2
0 0 SHM SHM+SPL OVX OVX+SPL SHM SHM+SPL OVX OVX+SPL
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a b
15 400 *
300 10 # Draft200 5
Uric acid (mg/dl) Uric # 100 Platelet / Lymphocyte / Platelet
0 0 SHM SHM+SPL OVX OVX+SPL SHM SHM+SPL OVX OVX+SPL
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84 * 63 Draft 42 *# GSK-3 (ng/ml) 21
0 SHM SHM+SPL OVX OVX+SPL
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800 65 * *
* 52 600 * * 39 # 400 # * 26
200 Aldosterone (pg/ml) 13 Corticosterone (pg/ml) Draft
0 0 SHM SHM+SPL OVX OVX+SPL SHM SHM+SPL OVX OVX+SPL
360 #
240 *
120 β 17 -Estradiol (pg/ml) -Estradiol 17
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Glucocorticoid E2 Aldosterone
MR
Draft GSK-3
Obesity/Insulin resistance
Hyperinsulinemia
Blockade CVDhttps://mc06.manuscriptcentral.com/cjpp-pubs T2DM