Effects of obesity induced by high-calorie diet and its treatment with exenatide on muscarinic acetylcholine receptors in rat hippocampus

Biochemical Pharmacology 169 (2019) 113630

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Effects of obesity induced by high-calorie diet and its treatment with exenatide on muscarinic acetylcholine receptors in rat hippocampus

T

Marcelo Florencio Passos Silvaa, Patricia Lucio Alvesa, Rafaela Fadoni Alpontia,b, ⁎ Paulo Flavio Silveiraa, Fernando Maurício Francis Abdallaa, a b

Laboratory of Pharmacology, Instituto Butantan, São Paulo, SP, Brazil Faculty of Medicine, Universidade do Oeste Paulista, Jau, SP, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Muscarinic acetylcholine receptors Obesity High-calorie diet Exenatide Hippocampus Inositol phosphate

Here, we described the effects of obesity induced by high-calorie diet and its treatment with exenatide, an antidiabetogenic and potential anti-obesogenic drug derived from the venom of the Gila monster Heloderma suspectum, on the affinity, density, subtypes and intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Male Wistar rats were divided into three groups: control (CT), obese induced by high-calorie diet (DIO) and DIO treated with exenatide (DIO + E). [3H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (KD) did not differ among CT, DIO and DIO + E, indicating that affinity is not affected by high-calorie diet or its treatment with exenatide. On the other hand, the density of mAChRs obtained in DIO animals was lower than that obtained from CT rats, and that DIO + E restored the density of mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M1 and M3 subtypes of DIO animals when compared with CT. Treatment with exenatide (DIO + E) restored the expression of the two subtypes similar to obtained from CT. On the other hand, the M2, M4 and M5 mAChR subtypes expression did not differ among CT, DIO and DIO + E. Carbacol caused a concentration-dependent increase in the accumulation of total [3H] inositol phosphate in CT, DIO and DIO + E. However, the magnitude of the maximal response to carbachol was lower in DIO when compared with those obtained from CT and DIO + E animals, which did not differ from each other. Our results indicate that obesity induced by highcalorie diet strongly influences the expression and intracellular signaling coupled to M1-M3 mAChR subtypes. The exenatide ameliorated these effects, suggesting an important role on hippocampal muscarinic cholinergic system. This action of obesity induced by high-calorie diet and its treatment with exenatide might be a key step mediating cellular events important for learning and memory.

1. Introduction Nowadays, obesity is considered the most prevalent nutritional disease around the world [1–3]. Obesity is a major risk factor for a variety of health conditions such as type 2 diabetes, dyslipidemia, hypertension and cardiovascular disorders collectively known as metabolic syndrome [4–6]. The experimental investigations of obesity have used genetically modified animals, or inductions of increased relative mass of fat pad through neonatal administration of monosodium glutamate [7] or high-calorie diet. Particularly, the high-calorie diet mimics the widespread global human consumption pattern of diets rich in sugars and fats [8,9], with remarkable features of comorbidity conditions that lead to obesity [10,11]. Glucagon-like peptide-1 (GLP-1) receptor agonists are a class of

⁎

incretin mimetic drugs that have been used for the treatment of type 2 diabetes mellitus and also hypothesized as beneficial in obesity [12–15]. The original prototypical drug in this class is the exenatide, a synthetic molecule of exendin-4 [16] isolated from Heloderma suspectum saliva. Exenatide is the first incretin mimetic peptide originated from animal toxin [17]. Regarding the biological activity, the insulinotropic potency of exenatide is similar to GLP-1 [16]. Exenatide is not a substrate of dipeptidyl peptidase IV, which gives it longer half-life and long-acting antihyperglycemic effect [17]. Furthermore, GLP-1 and derivative molecules cross the blood-brain-barrier when administered peripherally [18,19]. GLP-1 receptors are expressed within the neocortex and hippocampal regions of the brain, which are key areas responsible for modulating learning and memory processes [20–22]. Indeed, mice with targeted deletion of the GLP-1 receptor exhibit

Corresponding author. Laboratory of Pharmacology, Instituto Butantan, Av. Vital Brasil, 1500, CEP 05503-900 São Paulo, Brazil. E-mail address: [email protected] (F.M.F. Abdalla).

https://doi.org/10.1016/j.bcp.2019.113630 Received 11 July 2019; Accepted 30 August 2019 Available online 03 September 2019 0006-2952/ © 2019 Elsevier Inc. All rights reserved.

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2. Material and methods

demonstrable impairments in synaptic plasticity and cognitive performance whereas over-expression of GLP-1 in the hippocampus enhanced spatial learning [23,24]. In addition, GLP-1 has neuroprotective properties, protecting neurons from kainite-induced neurotoxicity and reducing hippocampal neuronal apoptosis [24,25]. A study from Gault and Hölscher [26] has shown that GLP-1 directly modulates neurotransmitter release and long-term potentiation (LTP) formation, and protects synapses from detrimental effects of β-amyloid fragments on LTP formation. In streptozotocin-treated rats simulating Alzheimer's disease, Solmaz et al. [27] showed that the exenatide was able to prevent the reduction of hippocampal neurons and suppress the inflammatory response, indicating that exenatide play an important role in cognitive processes. There are few studies that directly indicate the stimulatory effect of exenatide on cholinergic activity in the central nervous system. For instance, it has been found that both GLP-1 and exendin-4 prominently reduced ibotenic acid-induced depletion of choline acetyltransferase immunoreactivity in basal forebrain cholinergic neurons in rats [28]. Özyazgan et al. [29] reported that administration of streptozotocindiabetic rats with exenatide significantly normalized the reduction in acetylcholine-stimulated relaxations of aortic strips compared with normal control group. In another study, it has been suggested that exendin-4 markedly prevented the attenuation of acetylcholine-induced endothelium-dependent in diabetic rats [30]. Li et al. [31] showed in their study that exendin-4 elevated choline acetyltransferase activity in experimental model of amyotrophic lateral sclerosis. In addition, the administration of exenatide also preserved the activity of the choline acetyltransferase and improved memory in hippocampus from streptozotocin-treated rats simulating Alzheimer's disease, Solmaz et al. [27], suggesting that the effects of exenatide on hippocampal cholinergic neurotransmitter may be also a mechanism controlling cognitive processes. Muscarinic acetylcholine receptors (mAChRs) mediate a wide range of functions of the parasympathetic nervous system both centrally and peripherally. Different experimental approaches have shown that mAChRs are present in all organs, tissues, or cell types [32]. The muscarinic actions of acetylcholine are mediated by five distinct mAChR subtypes (M1 to M5) [33–35]. The five mAChR subtypes are expressed throughout the brain and play a role in a wide array of functional processes such as learning, memory, attention and sensorimotor processing [35–40]. The M1, M3, and M5 subtypes couple primarily to phopholipase C-mediated phosphoinositide hydrolysis. On the other hand, the M2 and M4 subtypes couple primarily to adenylyl cyclase inhibition [41]. Experimental studies has also been shown that obesity-associated with hypercaloric diet impair learning and memory in rodents [42–49], suggesting a strong association between obesity and cognitive dysfunction. However, the mechanisms of how the obesity induced by high-calorie diet affects memory are not understood fully. Potential mechanisms that may mediate the detrimental effect of obesity on cognition include at the level of the central nervous system (CNS), disruption of the blood brain barrier, central inflammation resulting from elevated pro-inflammatory cytokines and changes in membrane fluidity [42,44,50]. However, here we are the first to address the mechanisms underlying the effects of obesity induced by high-calorie diet and its treatment with exenatide on intracellular signaling linked to activation of mAChRs in the hippocampus of rats. Thus, the aim of this study was to investigate the effects of obesity induced by high-calorie diet without treatment and treated with exenatide on affinity, density, subtypes and production of inositol phosphate induced by stimulation of mAChRs.

2.1. Animals and treatments The conduct and procedures involving animal experiments were approved by the Butantan Institute Committee for Ethics in Animal Experiments (License number CEUAIB 22381003/17) in compliance with the recommendations of the National Council for the Control of Animal Experimentation of Brazil (CONCEA). All efforts were made to minimize suffering. 72–75 day old Wistar male rats, coming from the Central Animal Laboratory of the Instituto Butantan, were housed in a polypropylene box (inside length × width × height = 56 cm × 35 cm × 19 cm) within a ventilated container (Alesco Ind. Com Ltda, Monte Mor, Brazil) under controlled temperature (23 ± 2 °C), relative humidity (65 ± 1%) and 12 h light: 12 h darkness cycle (lights on at 6:00 a.m.) and subdivided into 2 groups. A group had access to hyperlipidic food and allowed to feed ad libitum: PragSoluções Biociencias, Jau, Brazil, containing the following composition: 14% protein (w/w), 42% carbohydrate (w/w), 32% lipids (w/w), 5% fiber (w/w), and 7% vitamins and minerals (w/w) (total of 5.2 kcal/g) [51]. These animals were allowed to drink only 30% sucrose in distilled water (1.2 kcal/mL) ad libitum [52]. Another group received normocaloric diet presenting the following composition: 22% protein (w/w), 55% carbohydrate (w/w), 4% lipids (w/w), 9% fiber (w/w), and 10% vitamins and minerals (w/ w) (total of 3 kcal/g) (Nuvilab CR-11, Sogorb, São Paulo, Brazil) and were allowed to feed and to drink water ad libitum. 142–145 day old rats with 20% overweight relative to the normal for age and lineage [51] were selected from the first group as obese animals (DIO) (10weeks DIO exposure). 142–145 day old rats with normal weight (429–435 g) were selected from the second group as control animals (CT). The animals not included in the experiments were euthanized under anesthesia by decapitation under anesthesia with intraperitoneal administration of 2 ml per kg body mass of a 3:2:4 mixture of ketamine hydrochloride solution (100 mg/mL), hydrochloride xylazine solution (100 mg/mL), and distilled water. DIO remained without treatment during the subsequent 20 days; DIO + E received a daily subcutaneous bolus injection of exenatide in saline 0.9% (10 µg/kg body mass), at a maximum volume of 200 ml, between 3:00–4:00 h of light period (9:00–10:00 a.m.), during the subsequent 20 days. Dose and duration of exenatide treatment were selected according to previous studies from humans [53,54], mice [55] and rats [56]. In another series of experiments for immunoprecipitation assays for detection of mAChR subtypes, CT animals from 17-week-old until 20week-old received a daily subcutaneous bolus injection of exenatide as described above, during the subsequent 20 days (E). The body mass gain of E in the end of evaluation (20 weeks old) decreased when compared with those obtained from CT (20 week-old) (respectively 388.8 ± 3.3, n = 10 and 432.0 ± 3.0, n = 64; P < 0.05; ANOVA, Student's t-test), in agreement with previous studies [57]. 2.2. Body mass and food intake Body mass progression of CT, DIO and DIO + E animals from 10week-old until 20-week-old was monitored. Food intake were also monitored from 10 weeks until 20 weeks of age in all experimental groups, respectively by measurements of weight (g) of the initial and remaining contents in their containers for food, every 48 h. Values obtained were extrapolated for one week. 2.3. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) Glycemia (mg/dL) was measured in the second drop of blood taken from the tip of the tail, using the portable glucose monitor Accu-Chek Advantage (Roche Diagnostics, Mannheim, Germany). In order to perform OGTT, animals were submitted to a 15-h fasting period and then 2

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the binding reaction was stopped by cooled on ice and rapid filtration through GF/B glass fiber under vacuum. The filters were washed three times with the ice-cold buffer, partially dried under vacuum and placed in scintillation vials containing OptiPhase HiSafe 3. The amount of radioactivity was determined in a scintillation β-counter. In another series of experiments, membrane preparation (100 μg) (in duplicate) was incubated with 1.5 nM [3H]QNB in the absence (total binding) and presence of 1 µM atropine (nonspecific binding) for 1 h at 30 °C. [3H]QNB-receptors were solubilized with digitonin and sodium deoxycholate [62–64] in 25 mM Tris-HCl, pH 7.4 (containing 5 mM MgCl2, 0.1% sodium deoxycholate, 1 mM EDTA and 1 mM PMSF) for 1 h at 4 °C. After that, 0.1% digitonin was added. The sample was incubated for 15 min at 4 °C and centrifuged at 15,000 g for 20 min at 4 °C. The supernatant, containing the solubilized receptors, was collected and the amount of radioactivity was determined. The supernatant, containing the solubilized receptors, was also immunoprecipitated by subtype-specific antibodies. The supernatant (100 µl), containing solubilized receptors, was incubated with 0.5 µg of polyclonal antibodies raised against goat mAChR subtypes [M1 (C-20, sc 7470), M2 (C-18, sc 7472), M3 (C-20, sc 7474) M4 (C-19, sc 7476) and M5 (C-20, sc 7478), Santa Cruz Biotechnology, CA, USA] (specific) or with IgG (nonspecific) (Sigma Co, MO, USA) in 25 mM Tris-HCl, pH 7.4 (containing 5 mM MgCl2, 1 mM EDTA and 1 mM PMSF) for 4 h at 4 °C. After this incubation, 20 µl of Pansorbin (Calbiochem, CA, USA) were added and incubated under agitation for 1 h at 4 °C and then centrifuged at 15,000 g for 10 min [62,63]. The pellet was washed with 200 µl Tris-HCl, pH 7.4 (containing 5 mM MgCl2, 1 mM EDTA and 1 mM PMSF) and centrifuged again at 15,000 g for 10 min. The amount of radioactivity was determined in 50 µl Pansorbin deposit ([3H]QNBsubtype-specific antibodies) (total) or Pansorbin deposit ([3H]QNB-IgG) (nonspecific). The specific immunoprecipitation (difference between the total and nonspecific) were determined and the results were expressed as fmol/mg protein [62]. To validate the immunoprecipitation assays, in rat hippocampus from control (CT, normocaloric feed), the percentage of specific immunoprecipitation for each mAChR subtype was also calculated by dividing the amount of [3H]QNB count in the Pansorbin pellet by the whole count in the total supernatant and Pansorbin pellet [62]. Our results confirmed that the population of M1 mAChRs is not the only one in the hippocampus, but their proportion in the total amount of receptors is very high (68.1 ± 6.1%, n = 4). These results also showed that on the protein level the amount of M2 in the hippocampus is higher than the amount of M3, M4 and M5 mAChRs (respectively, 31.5 ± 6.4, n = 5; 11.2 ± 4.7, n = 6; 10.5 ± 5.8, n = 4 and 0.6 ± 0.4%, n = 3) (P < 0.05; ANOVA, Newman-Keuls test). It is important to emphasize that our immunoprecipitation studies are in accordance with other reports in the literature. In hippocampus of male rats immunohistochemistry studies also indicated a predominance of M1 subtype (55%) and low expression of M2 (17%), M3 (10%) and M4 (15%) [65]. Binding studies using [3H]methyl-scopolamine showed 69% expression of M1, 30% of M2, 17% of M3, 9% of M4 and 1% of M5 receptors in the hippocampus of male mice [66]. Similarly, immunoprecipitation studies in hippocampus obtained from female rats in proestrous phase revealed 61% expression of M1, 23% of M2, 8% of M3, 6% of M4 and 0.6% of M5 receptors [63].

glycemia was measured at 0 time (basal). Subsequently, animals received 2 g glucose (Synth, Sao Paulo, Brazil) in water/kg body mass, in a maximum volume of 1 ml, via oral by gavage, and the glycemia was measured at 15, 30, 60, 120 and 180 min. The areas under the timecourse curve of glycemia (AUC) were calculated [58]. In order to perform ITT, animals were submitted to a 12-h fasting period and then glycemia at 0 time (basal) was measured. Subsequently, animals received 0.75 UI insulin (Eli Lilly and Co., Indianapolis, USA) in saline/kg body mass, in a maximum volume of 0.75 ml, via intraperitoneal, and the glycemia was measured at 15, 30 and 60 min and then converted into natural logarithm (Ln); the slope of time-course curve of Ln glucose was calculated using linear regression [time × Ln (glucose)] and multiplied by 100 to obtain the constant rate of glucose decay per minute (KITT) [59]. 2.4. [3H]Quinuclidinyl benzilate ([3H]QNB) binding assay Hippocampus membrane preparation, obtained from 6 animals for each experiment, was prepared as described by Cardoso et al. [60]. Briefly, entire hippocampus was isolated from all experimental groups, minced and homogenized in 25 mM Tris-HCl, pH 7.4 (containing 0.3 M sucrose, 5 mM MgCl2, 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride) with a Ultra-Turrax homogenizer (T-25, Ika Labortechnik, Staufen, Germany). The homogenate was centrifuged at 1000 g for 10 min. The supernatant was filtered through two layers of gauze and then centrifuged at 100,000 g for 60 min. At the final pellet (drop 100,000 g) was resuspended in 1 ml of 25 mM Tris-HCl, pH 7.4 (containing 5 mM MgCl2, 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride), using a Dounce homogenizer and stored at −80 °C. All procedures were carried out at 4 °C, and all solutions contained freshly added 1 mM phenylmethylsulfonyl fluoride to inhibit proteolysis. Protein concentration was determined with a BioRad protein assay, using BSA as standard (Bio Rad Laboratories, Hercules, CA). Saturation-binding experiments were performed as previously described [60]. Briefly, hippocampus membrane preparation (100 µg protein/ml) was incubated with 0.05–1.8 nM [3H]QNB (specific activity 44.0 Ci/mmol; New England Nuclear, Boston, MA, USA) in the absence (total binding) and presence (nonspecific binding) of 1 µM atropine for 1 h at 30 °C. After incubation, the binding reaction was stopped by cooled on ice and rapid filtration through GF/B glass fiber filter (Whatman International Ltd, Maidstone, UK) under vacuum. The filters were washed three times with the ice-cold buffer, partially dried under vacuum and placed in scintillation vials containing OptiPhase HiSafe 3 (Perkin Elmer, Loughborough Leics., UK). The amount of radioactivity was determined in a scintillation β-counter (LS 6500 IC, Beckmann, USA). Specific binding was calculated as the difference between total and nonspecific binding. The nonspecific binding, near the KD value, was about 10% of the [3H]QNB total binding. Saturation binding data were analyzed using a weighted nonlinear least-squares interactive curvefitting program GraphPad Prism (GraphPad Prism Software Inc., San Diego, CA, USA). A mathematical model for one or two binding sites was applied. The equilibrium dissociation constant (KD) and the binding capacity (Bmax) were determined from Scatchard plot [61]. 2.5. Immunoprecipitation assays for detection of mAChR subtypes

2.6. Measurement of total [3H]inositol phosphate Receptor labeling, solubilization and immunoprecipitation: Muscarinic acetylcholine receptors present in the membranes were radiolabeled with 1.5 nM of [3H]QNB, a subtype-nonselective antagonist with a high affinity and very slow dissociation. Briefly, membrane preparation (100 μg) (in duplicate) was incubated with 1.5 nM [3H] QNB (maximum binding obtained in saturation binding experiments) in the absence (total binding) and presence of 1 µM atropine (nonspecific binding) for 1 h at 30 °C. The specific binding was calculated as the difference between the total and nonspecific binding. After incubation,

Hippocampi were isolated from CT, DIO and DIO + E rats, and washed with a nutrient solution of the following composition (mM): NaCl 118.00; KCl 4.78; CaCl2 2.43; MgSO4 1.16; NaHCO3 23.80; KH2PO4 1.17; glucose 2.92 (pH 7.4). Hippocampus slices (100 mg of tissue) were allowed to equilibrate for 10 min in nutrient solution at 37 °C under constant shaking. The slices were incubated for 40 min with 1 μCi myo[3H]inositol (specific activity 47.0 Ci/mmol; New England Nuclear, Boston, MA, USA), and for an additional 30 min with 10 mM 3

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A

lithium chloride with myo[3H]inositol. Tissues were then incubated in the absence (basal level) and presence of carbachol (10−7 to 10−3 M) (Sigma) for 40 min. Tissues were washed three times with nutrient solution, transferred to 2 ml of methanol:chloroform (2:1 v/v) at 4 °C and homogenized with a Ultra-Turrax T25 homogenizer at 9500 rpm. Chloroform (0.62 ml) and H2O (0.93 ml) were added to the homogenate, and the solution was centrifuged for 10 min at 2000 g and 4 °C to separate the aqueous and organic phases [67,68]. Total [3H]inositol phosphate was measured as previously described by Ascoli et al. [69] with the following modification. The aqueous layer was mixed with 1 ml anion-exchange resin (Dowex AG-X8, formate form, 200–400 mesh), allowed to equilibrate for 30 min at room temperature, and centrifugated at 1000 g for 5 min at 4 °C. The resin was then washed sequentially with myo-inositol (4 ml) and 5 mM sodium tetraborate/60 mM sodium formate (2 ml). The resin was incubated for 30 min at room temperature with 2 ml of 0.1 M formic acid/1 M ammonium formate. The total [3H]inositol phosphate was eluted and placed in scintillation vials containing OptiPhase HiSafe 3. The amount of radioactivity was determined in scintillation βcounter. Total [3H] inositol phosphate was expressed as % above basal level.

600

Body mass (g)

500 400

#

#

#

#

#

* *

*

*

* *, **

* **

300 200 100

CT DIO DIO+E

0

10 11 12 13 14 15 16 17 18 19 20

Age (weeks)

B 1000

Food intake (g)

800

2.7. Statistical analysis Data were expressed as mean ± SEM. Data were analyzed by ANOVA followed by Newman-Keuls test for multiple comparisons or by the two-tailed Student's t-test to compare a response between two groups [70]. P values < 0.05 were accepted as significant.

600

200 0

3. Results

*

#

#

400

* * # #

#

CT DIO DIO+E

*,**

*,**

*,**

10 11 12 13 14 15 16 17 18 19 20

3.1. Body mass and food intake

Age (weeks)

The comparisons of body mass and food intake among CT, DIO and DIO + E are shown in Fig. 1. The body mass gain (Fig. 1A) was higher in DIO than in CT between 12 and 20 weeks old (p < 0.05; ANOVA). 19-week-old DIO + E presented lower body mass gain than DIO (P < 0.05; ANOVA, Newman-Keuls test). However, the end of evaluation (20 weeks old) DIO + E presented similar body mass gain than DIO (p > 0.05; ANOVA). The body mass gain was higher in DIO + E than in CT between 17 until 19 weeks old (p < 0.05; ANOVA, Newman-Keuls test). The body mass gain of CT, DIO and DIO + E in the 20 week-old was 432.0 ± 3.0, n = 64; 538.3 ± 6.5, n = 53 and 465.2 ± 4.7 g, n = 31, respectively. In DIO, the food intake (Fig. 1B) was lower in the 10, 14 and 16 weeks old, when compared with CT (p < 0.05; ANOVA, Student's ttest). Food intake of DIO and DIO + E in the 17-week old was similar than in CT, but from 18-week-old until the end of evaluation (20 weeks old) food intake was lower in DIO and DIO + E when compared with CT. Furthermore, the food intake was lower in DIO + E than those obtained from DIO animals (p < 0.05; ANOVA, Newman-Keuls test).

Fig. 1. Body mass gain (A) and food intake (B) of healthy (CT ●) (n = 64), obese induced by high-calorie diet without treatment (DIO ■) (n = 53) and treated with exenatide (DIO-E ♦) (n = 31) from 10-week-old until 20- weekold. Values are mean ± S.E.M. The line indicates the week that treatment with exenatide starts. From 10-week-old until 16-week-old: #Indicates a statistically significant difference from CT in the same week (p < 0.05; ANOVA, Student's t-test). From 17-week-old until 20-week-old: *Indicates a statistically significant difference from CT in the same week (P < 0.05; ANOVA, Newman-Keuls test); **Indicates a statistically significant difference from DIO in the same week (P < 0.05; ANOVA, Newman-Keuls test).

presented higher glycemia than CT. However, in DIO at 9, 12, 15 min, ITT was similar than CT (p < 0.05; ANOVA, Newman-Keuls test). KITT was higher in DIO + E (1.12 ± 0.001%/min, n = 6) than in DIO (0.79 ± 0.002%/min, n = 6), but it was lower than in CT (2.02 ± 0.002%/min, n = 6) (p < 0.05; ANOVA, Newman-Keuls test).

3.2. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT)

3.3. [3H]QNB binding in the rat hippocampus

Fig. 2 shows the comparisons of the areas under the time-course curve of glycemia (AUC) in glucose tolerance test (OGTT), and time course of glycemia and the decay of glycemia per minute in function of insulin administration (KITT) in insulin tolerance test (ITT) among CT, DIO and DIO + E groups. During OGTT (Fig. 2A), DIO and DIO + E presented higher glycemia at 0, 120 and 180 min and higher area under the glycemic curve (respectively 31,219 ± 3094, n = 6 and 30,325 ± 1569, n = 6) than CT (22,565 ± 1446, n = 6), being similar between DIO and DIO + E (p < 0.05; ANOVA, Newman-Keuls test). During ITT (Fig. 2B), at 0, 3, 6, 30 and 60 min, DIO and DIO + E

The binding of [3H]QNB to hippocampus membranes from all experimental groups (CT, DIO and DIO + E) was specific and saturable. Scatchard analysis of specific binding fitted best a one-site model. An analysis of three experiments, performed in duplicate, yielded dissociation constant (KD) and maximum number of binding sites (Bmax) summarized in Table 1. The KD values did not differ among CT, DIO and DIO + E (p > 0.05; ANOVA) (Fig. 3; Table 1). On the other hand, DIO induced a decrease in the Bmax in the hippocampus when compared to those obtained from CT. The exenatide treatment reversed the effect on Bmax similar to those obtained from CT (Fig. 3; Table 1) (p < 0.05; ANOVA, 4

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Fig. 2. Time-course of glycemia and area under the glycemic curve (AUC) in glucose tolerance test (A) and time-course of glycemia and KITT in insulin tolerance test (B) of healthy (CT ●) (n = 6), obese induced by high-calorie diet without treatment (DIO ■) (n = 6) and treated with exenatide (DIO-E ♦) (n = 6). Data are mean ± S.E.M. *Indicates a statistically significant difference from CT (P < 0.05; ANOVA, Newman-Keuls test); **Indicates a statistically significant difference from CT or DIO (P < 0.05; ANOVA, Newman-Keuls test).

changed the M1 (4.49 ± 0.24 fmol/mg protein, n = 3) subtype in the hippocampus when compared to those observed from DIO (P < 0.05; ANOVA, Newman-Keuls test). However, DIO + E reversed the effect on the M3 (2.20 ± 0.48 fmol/mg protein, n = 3) subtype in the hippocampus similar to those obtained from CT and E (p < 0.05; ANOVA, Newman-Keuls test). On the other hand, the M2, M4 and M5 mAChR subtypes expression did not differ among CT, DIO, DIO + E and E (p > 0.05; ANOVA).

Table 1 [3H]QNB saturation binding parameters in hippocampus membranes from healthy rats (CT), obese induced by high-calorie diet without treatment (DIO) and treated with exenatide (DIO + E). Experimental Groups

KD (nM)

Bmax (fmol/mg protein)

CT DIO DIO + E

0.14 ± 0.06 (n = 3) 0.18 ± 0.04 (n = 4) 0.44 ± 0.26 (n = 3)

184.83 ± 12.40 (n = 3) 62.20 ± 12.07* (n = 4) 194.0 ± 21.38 (n = 3)

3.5. Effects of carbachol on total [3H]inositol phosphate accumulation

Data are expressed as mean ± S.E.M. of number of experiment (n) in parenthesis, performed in duplicate. *Indicates a statistically significant difference from CT or DIO + E (P < 0.05; ANOVA, Newman-Keuls test).

CT, DIO and DIO + E did not change the basal level of the total [3H] inositol phosphate in hippocampus (2.19 ± 0.13, n = 7; 2.31 ± 0.27, n = 5 and 2.42 ± 0.33 dpm/mg tissue, n = 3, respectively) (P > 0.05; ANOVA) (Fig. 5A). The cholinergic agonist carbachol (CCh, 10−7M to 10−3M) caused a dose-dependent increase of total hippocampal [3H]inositol phosphate in all experimental groups (Fig. 5B). Maximum inositol phosphate accumulation was obtained with 10−5 M CCh. DIO decreased the response to the 10−5 M CCh (7.58 ± 4.92% above basal, n = 5) when compared to CT (28.96 ± 4.99 above basal, n = 5). Treatment of DIO with exenatide (DIO + E) reverted this effect (25.03 ± 8.09% above basal, n = 5). (P < 0.05; ANOVA, Newman-Keuls test).

Newman-Keuls test). 3.4. Expression of muscarinic receptor subtypes in the rat hippocampus Subtype-specific antibodies to mAChRs were used for quantifying these receptors in the hippocampus from all experimental groups (CT, DIO and DIO + E) (Fig. 4). Moreover, an additional group was also included as a control of DIO + E (E; CT animals treated with exenatide). DIO induced a decrease of the M1 (0.66 ± 0.27 fmol/mg protein, n = 7) and M3 (0.24 ± 0.14 fmol/mg protein, n = 7) mAChR subtypes expression in the hippocampus when compared to those obtained from control rats (CT) (8.91 ± 0.73, n = 6 and 2.84 ± 0.93 fmol/mg protein, n = 8, respectively M1 and M3) and E (8.61 ± 1.95, n = 4 and 3.21 ± 1.39 fmol/mg protein, n = 4, respectively M1 and M3) (P < 0.05; ANOVA, Newman-Keuls test). Each mAChR subtype did not change when compared between CT and E animals (P > 0.05; ANOVA). Treatment of DIO animals with exenatide (DIO + E) slightly

4. Discussion The results of the present study show, for the first time, that mAChRs function is affected by obesity induced by high-calorie diet (DIO). Moreover, the treatment of obese animals with exenatide (DIO + E) may be important to regulate the function of mAChRs. 5

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Fig. 3. Saturation curves (left panel) and Scatchard plot (right panel) of [3H]QNB bound to hippocampus membranes from healthy rats (CT) (A), obese induced by high-calorie diet without treatment (DIO) (B) and treated with exenatide (DIO + E) (C). Specific binding (●) is the difference between total binding (♦) and nonspecific binding (■). Scatchard plot derived from the same data of specific binding. Results are representative of n = 3–4, performed in duplicate.

induce glycemic dysregulation in addition to obesity in rats. Furthermore, the usual therapeutic dose of exenatide in humans [53,54] is antiobesogenic in diet induced obesity, indicating that the agonist of GLP1R is promising anti-obesogenic drug in the obesity. In hippocampus, acetylcholine and other neurotransmitters play an important role in cognitive processes [32,41]. In order to understand the relationship between obesity induced by high-calorie diet and mAChRs, we studied the effect of high-calorie diet (DIO) as well as its treatment with exenatide (DIO + E) on affinity, density, subtypes and production of inositol phosphate induced by stimulation of mAChRs in hippocampus from rats. Our results with [3H]QNB saturation binding studies indicated the presence of one class of high-affinity sites for QNB in the hippocampus from all experimental groups. The KD values obtained to mAChRs in rat hippocampus were similar among control, DIO and DIO + E, indicating that affinity is not affected by high-calorie diet

Obesity induced by high-calorie diet without treatment and treated with exenatide was previously studied in our laboratory [56]. Highcalorie diet was characterized by glucose intolerance, increased body mass, Lee index, fluid intake, mass of retroperitoneal and periepididymal fat pads, glycemia, glycated hemoglobin (HbA1c), triglycerides, very low density lipoprotein (VLDL) and total cholesterol, as well as decreased food intake and KITT. Exenatide restored glycemia, HbA1c, triglycerides, VLDL, total cholesterol and body mass, and it also ameliorated food and fluid intake, KITT and mass of retroperitoneal fat pad in DIO [56]. The present study glucose intolerance, increased body mass, glycemia, as well as decreased food intake and KITT was also obtained in high-calorie diet. On the other hand, exenatide restored glycemia and body mass, and it also ameliorated food intake and KITT in DIO. Taken together these results are in accordance with Alves et al. [56]. Thus, our studies collectively reflect that the high-calorie diet 6

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A 4

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Fig. 5. The basal levels (A) and concentration-effect curves of carbachol (B) on total [3H]inositol phosphate accumulation in hippocampus obtained from of healthy (CT●), obese induced by high-calorie diet without treatment (DIO ■) and treated with exenatide (DIO-E ♦). Each point and vertical line represent the means ± S.E.M. of n = 3–6. *Indicates a statistically significant difference from CT or DIO + E (P < 0.05; ANOVA, Newman-Keuls test).

E

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or its treatment with exenatide. Interestingly, a decrease in the Bmax in hippocampus from DIO animals was found, suggesting that obesity induced by high-calorie diet regulates the density of total mAChRs. To our knowledge, this is the first study demonstrating that total mAChRs density is affected by 10-weeks DIO exposure in hippocampus. The exact mechanisms underlying the effects of obesity on down-regulation of mAChRs in rat hippocampus were not explored in the present study which needs to be better investigated. Studies have demonstrated the beneficial utility of the GLP-1 receptor signaling pathway in preclinical models of several CNS related neurological disorders such as Alzheimer's disease, Parkinson's disease, stroke, amyotrophic lateral sclerosis and Huntington's disease [71–75]. Due to the apparent benefits of GLP-1 receptor signaling in quite diverse neurodegenerative conditions, we examined the potential benefits from the peripheral administration of a GLP-1 receptor agonist, exenatide which readily crosses the blood–brain barrier [18,19], in DIO + E animals. Notably, treatment of DIO with exenatide reversed the effect on total mAChRs density since was similar to those obtained from control rats, suggesting that exenatide also plays an important role on muscarinic cholinergic neurotransmitters in the hippocampus. The GLP-1 receptor is expressed in several regions of rodent and human brain, including the hippocampus [20–22]. Moreover, in a beta cell line model (BRIN BD11) an important functional cooperation between the cholinergic neurotransmitters acetylcholine and the incretin hormone GLP-1 on insulin secretion mediated through the M3 mAChR subtype was demonstrated [76], suggesting that exenatide may modulate the expression of mAChRs. Thus, the efficacy of GLP-1 receptor observed here is in line with literature describing exenatide brain entry [18], and

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Fig. 4. M1 to M5 muscarinic acetylcholine receptor subtypes expression in hippocampus obtained from healthy rats (CT), obese induced by high-calorie diet without treatment (DIO) and treated with exenatide (DIO + E), and CT animals treated with exenatide (E). Data are mean ± S.E.M. of n = 3–7. *Indicates a statistically significant difference from CT, DIO + E or E (P < 0.05; ANOVA, Newman-Keuls test); **Indicates a statistically significant difference from CT, DIO or E (P < 0.05; ANOVA, Newman-Keuls test).

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Acknowledgements

GLP-1 receptor-mediated actions in various CNS neurological conditions [75,77–79]. Subtype-specific antibodies to mAChRs (targeted to specific peptide sequences of all five subtypes) have been developed [80–86] and used for quantifying mAChRs by immunoprecipitation in a number of tissues in various species. In general, immunoprecipitation experiments are more sensitive than Western blot. Therefore, we employed this method to study the effects of obesity induced by high-calorie diet and its treatment with exenatide on expression of mAChR subtypes in order to detect the subtype(s) involved in the regulation previously observed in [3H]QNB saturation binding assays. Obesity induced by high-calorie diet (DIO) decreased the M1 and M3 mAChR subtypes expression. Treatment of DIO animals with exenatide (DIO + E) restored the expression of these two subtypes. Although, for M1 subtype, the effect was partial since it was still significantly different from the control, suggesting that the dose and/or time of treatment with exenatide may be involved in this effect. On the other hand, M2, M4 and M5 mAChR subtypes expression did not differ among CT, DIO, DIO + E and E. Taken together, these results indicate a differential modulation of M1 and M3 mAChR subtypes from obese rats induced by high-calorie diet. It is important to emphasize that each mAChR subtype did not change when compared between CT and E animals. Thus, our results also suggest that the treatment of obese animals with exenatide may be important to regulate the expression of mAChR subtypes. Further experimental approaches will be important to clarify the regulation of M1 and M3 mAChR subtypes and the functional significance. Our results confirmed that the population of M1 mAChRs is not the only one in the hippocampus, but their proportion in the total amount of receptors is very high. These results also showed that on the protein level the amount of M2 in the hippocampus is higher than the amount of M3, M4 and M5 mAChRs. It is known that M1 subtype in the cerebral cortex and hippocampus may play an important role in the higher cognitive processes such as learning and memory [32,41,87]. The predominant existence of M1 subtype in the hippocampus may provide further convincing evidence that M1 subtype is significantly involved in the higher cognitive function. Carbachol caused a dose dependent increase of total [3H]inositol phosphate in hippocampus from CT, DIO and DIO + E. The magnitude of the response to 10−5M carbachol was higher in hippocampus from CT and DIO + E rats than in hippocampus from DIO animals. These studies suggest that the obesity induced by high-calorie diet is important to regulate the number and function of mAChRs in hippocampus. Moreover, we can not exclude the possibility that a critical period with regard to the initiation of exenatide treatment is important to protect cognitive function, as previously reported [20,21,23,24,27,71–75,88–91]. Thus, the modulation on total [3H]inositol phosphate accumulation induced by high-calorie diet and its treatment with exenatide in hippocampus from rats may play a role in the hippocampal function. The activation of phosphoinositide hydrolysis contributes to various neuronal processes such as the changes in synaptic plasticity that underlies learning and memory [92–95]. In conclusion, the modulation of M1 and M3 mAChR subtypes expression and, consequently, the intracellular signaling pathways (PLCmediated phosphoinositide hydrolysis) points out for physiological significance especially on cognitive functions. These findings provide new insight into how obesity induced by high-calorie diet can influence the muscarinic cholinergic system in hippocampus. Furthermore, treatment of obesity with exenatide may be also potential therapeutic targets whose evaluation has been started with the present study.

This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant 2016/24258-5). M.F.P.S. and P.L.A. were recipients of a Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) fellowship. References [1] V.S. Malik, W.C. Willett, F.B. Hu, Global obesity: trends, risk factors and policy implications, Nat. Rev. Endocrinol. 9 (1) (2013) 13–27. [2] C.J. Lavie, A. De Schutter, P. Parto, E. Jahangir, P. Kokkinos, F.B. Ortega, R. Arena, R.V. Milani, Obesity and prevalence of cardiovascular diseases and prognosis-the obesity paradox updated, Prog. Cardiovasc. Dis. 58 (5) (2016) 537–547. [3] A. Engin, The definition and prevalence of obesity and metabolic syndrome, Adv. Exp. Med. Biol. 960 (2017) 1–17. [4] A. Tchernof, J.P. Despres, Pathophysiology of human visceral obesity: an update, Physiol. Rev. 93 (1) (2013) 359–404. 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Declaration of Competing Interest The authors declare that there is no conflict of interests regarding the publication of this paper.

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