Ex vivo and in silico evaluations of (E)-5-((benzo[d]thiazol-2-ylimino)(methylthio)methylamino)-2-hydroxybenzoic acid as a β3-adrenoreceptor agonist exerting anti-obesity, anti-inflammatory and hepatoprotective effects on Zucker rats

TL;DR

A new compound was tested in obese rats and found to reduce weight, inflammation, and liver damage by activating a specific receptor.

Contribution

The study demonstrates a novel β3-adrenoreceptor agonist with anti-obesity, anti-inflammatory, and hepatoprotective effects in Zucker rats.

Findings

C1 significantly reduced bodyweight and daily growth in obese Zucker rats.

C1 activated β3-ADR and stimulated thermogenesis via UCP-1 expression.

C1 decreased free fatty acids and tumor necrosis factor alpha levels.

Abstract

Obesity increases oxidative stress and inflammation and thereby promotes liver damage and metabolic disorders, such as type 2 diabetes. Although exist anti-obesity treatments with employing anorexic drugs, antioxidants, and β3-adrenergic receptor (β3-ADR) agonists, their use adversely effects human health. Herein, the agonistic effect of 5-(((benzo[d]thiazol-2-ilimino)(methylthio)methyl)amino)-2-hydroxybenzoic acid (C1) on β3-ADRs as well as its hepatoprotective and anti-inflammatory properties are investigated in obese Zucker rats. When compared to clobenzorex, C1 (12 mg/Kg bodyweight per day) intragastrically administered for 28 days significantly reduced the bodyweight and daily growth of the animals (p < 0.0001), as it stimulated thermogenesis by activating the uncoupling protein 1 (UCP-1) (p = 0.0090), which was mediated by the positive expression of β3-ADR (p = 0.0149). C1-induced β3-ADR activation was attributed to the formation of conventional hydrogen bonds between the hydroxyl and secondary amine groups of C1 with ASP117, VAL121, and THR122 at the catalytic site on the receptor. In addition, C1 significantly decreased the levels of free fatty acid (p < 0.0001) and tumor necrosis factor alpha (p = 0.0073). Furthermore, C1 increased glutathione levels (p = 0.0245), and showed a tendency to decrease lipid oxidation and interleukin 6 levels. Therefore, these findings nevertheless suggested that C1 acted as a β3-ADR agonist and exerted hepatoprotective and anti-inflammatory effects on obese Zucker rats.

Figures

Chemical structure of C1.

Zucker rat attributes evaluated under different experimental conditions.(a) Bodyweight (g) growth curves of the saline- and vehicle-treated Zucker rats in the LRS (●), LRV (o), ORS (■), and ORV (□) groups plotted as functions of age (days), (b) average DRG rates of the C1, CLX, and vehicle-treated lean (LR) and obese (OR) rats, (c) cumulative food consumption of the rats in the LRS (●), ORV (□), ORCLX (▲), and ORC1 (♦) groups, and (d) survival rates (percentages) of the differently treated LR and OR. The lines represent the observed survival rates; the y-axis represents the survival proportion, and the x-axis represents the treatment time (28 days). ap < 0.0001 against the LRS group, bp < 0.0001 compared with the LRV group, cp < 0.0001 vs. the ORS group, and dp < 0.0001 relative to the ORV group. A comparison of the survival curves shows no significant differences were obtained over time using the Kaplan–Meier statistic and log-rank (Mantel–Cox) test.

Antioxidant and oxidant hepatic markers of Zucker rats under different treatments.(a) Average GSH and (b) TBARS levels. The numbers of samples in each group were LRS = 7, LRV = 7, ORS = 7, ORV = 5, ORCLX = 6, and ORC1 = 7. Each experiment was performed in triplicate; ap = 0.0245 against the OVR group.

Blood glucose and insulin levels of Zucker rats after 28 days of different treatments.(a) Average blood glucose and (b) plasmatic insulin levels of control and experimental groups. The numbers of samples in each group was: LRS = 7, LRV = 7, ORS = 7, ORV = 5, ORCLX = 6, and ORC1 = 7. Each experiment was performed in triplicate; ap = 0.0002 against LRS and bp = 0.0002.

Correlations among the plasmatic lipid profiles of Zucker rats after 28 days of different treatments.(a) TG and (b) FFA levels. The numbers of samples in each group were LRS = 7, LRV = 7, ORS = 7, ORV = 5, ORCLX = 6, and ORC1 = 7. Each experiment was performed in triplicate; ap < 0.0001 against LRS, bp < 0.0001 compared to LRV, cp < 0.0001 compared to ORS, dp < 0.0001 against ORV, and ep = 0.0002 against ORCLX.

Plasmatic cytokine levels of Zucker rats under different treatments.(a) TNF-α and (b) IL-6 levels. The numbers of samples in each group were LRV = 7, ORV = 5, ORCLX = 6, and ORC1 = 7. Each experiment was performed in triplicate; a,b,cp = 0.0073 against ORV, LRV, and ORCLX, respectively.

Relative mRNA expression levels.(a) β3-ADR and (b) and UCP-1 in the AT of Zucker rats under different treatments. The numbers of samples in each group were LRV = 7, ORV = 5, ORCLX = 6, and ORC1 = 7. Each experiment was performed in triplicate; ap = 0.0149 and bp = 0.0090 compared with LRV, ORV, and ORCLX.

Validation of the β3-ADR–epinephrine complex docking.(a) Superposition of the co-crystallized (pink) and redocked (green) complexes at the β3-ADR active site. (b) A 3D representation of epinephrine co-crystalized at the orthosteric site. (c) Epinephrine redocked using VMD at the receptor’s active site.

Representation of ligands docking at the β3-ADR active site.(a) Three-dimensional representations of the co-crystallized ligand epinephrine (pink), C1 (purple), and CLX (yellow) in the binding cleft of the adrenoreceptor (PDB ID: 9IJE) and 3D representations of (b) CLX and (c) C1 docked at the active site of the receptor, as modeled using VMD.

Full text

1. Introduction

Obesity is a chronic medical condition that is characterized by excessive accumulation of body fat, which can substantially adversely affect health [1]. Obesity has been linked to the development of nonalcoholic fatty liver disease (NAFLD) [2], which is characterized by lipid accumulation as triglycerides (TGs) in the liver. This pathology is described as an inflammatory process as lipids transform to free fatty acids (FFAs) via lipolysis, contributing to abnormally increased expressions of cytokines and adipokines, such as interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), IL-1, leptin, and resistin [3]. Increased cytokine expression promotes the overproduction of reactive oxygen species (ROSs), inhibiting cellular antioxidant enzymes and exacerbating oxidative stress (OS), which can damage proteins and lipids [4]. Lipid peroxidation (LPO) involves the production of lipid hydroperoxides mediated by polyunsaturated fatty acids during the breakdown and degradation of cell membranes, generating conjugated dienes and malondialdehyde (MDA) [5], which react with thiobarbituric acid (TBA) to form MDA-TBA2 adducts known as TBARSs (TBA-reactive substances), which are LPO indicators [6]. LPO can alter the functions of many types of cells and tissues (e.g., vascular endothelial cells, myocytes, and pancreatic beta cells) and the development of dyslipidemia, diabetes mellitus type 2 (T2DM), metabolic syndrome (MS), coronary heart disease, and hypertension [7].

Owing to the crucial role of OS in obesity development, the antioxidant defense system becomes important to reduce, delay, and potentially prevent ROS-induced damage. Owing to its role as a precursor for the synthesis of the endogenous antioxidant reduced glutathione (GSH), N-acetylcysteine is employed as an alternative antioxidant [8]. Similarly, polyphenols are another important group of compounds possessing antioxidant effects that can counteract generated OS. Polyphenols are characterized by hydroxyl group–substituted aromatic rings that can neutralize ROSs. However, as polyphenols possess limited bioavailability, their use is considered as unsafe and ineffective [9–11].

Anorexic drugs are utilized to counteract obesity and its comorbidities. Drugs such as sibutramine, fenfluramine, dexfenfluramine, methamphetamine, and clobenzorex (CLX) are sympathomimetic amines exerting anorexigenic effects mediated by activating serotonergic and noradrenergic pathways. Their main action mechanism involves reducing food intake and enhancing satiety by acting on the hypothalamus. Nevertheless, these drugs have been linked to severe side effects, such as nonfatal myocardial infarction and stroke, cardiac valvular insufficiency, and pulmonary hypertension, decreased motor activity and motor coordination and neurotoxicity, and a high incidence of adverse reactions. As these risks increase the potential for drug abuse, these drugs have been mostly withdrawn from the pharmaceutical market in some parts of the world [12,13].

As ~30% of the world’s population is affected by obesity and current treatments are inadequate for counteracting bodyweight gain–associated OS and inflammatory processes, the development of effective therapies is essential [14].

Because of its extensive presence in the inner mitochondrial membrane of brown adipose tissue (BAT) and its important role in energy expenditure by converting food energy to body heat, uncoupling protein 1 (UCP-1) has emerged as a promising target for obesity treatment. UCP-1 remains inactive and does not generate heat unless activated, as it is inhibited by purine nucleotides. However, because the activation of UCP-1 via beta-3 adrenergic receptors (β3-ADRs) enhances thermogenesis, targeting UCP-1 activation via β3-ADRs with synthetic and natural small molecules represents a promising strategy for addressing obesity. Mirabegron (YM178), solabegron (GW427353), ritobegron (KUC-7483), SR 58611, BRL 37344, CGP 12177, BRL 27344, CL 316243, and antioxidants have been considered as β3-ADR receptor agonists that can stimulate both cyclic adenosine monophosphate (cAMP) synthesis and UCP-1 mitochondrial activation because they interact within the receptor’s active site [15,16]. Reportedly, epinephrine also binds to the β3-ADR catalytic site, comprising ASP117, SER169, SER209, SER212, and PHE309 residues. Although these drugs are primarily used to treat an overactive bladder, they are linked to adverse effects and not truly effective β3-ADR agonist is currently available [17].

In this context, we demonstrated that 5-(((benzo[d]thiazol-2-ylimino)(methylthio)methyl)amino)-2-hydroxybenzoic acid (C1, Fig 1) exerted a hepatoprotective effect on the homogenized livers of acetaminophen-overdosed CD-1 mice, as it inhibits LPO and cytochrome P450 complex (CYP450) and increase endogenous antioxidant synthesis (GSH and glutathione reductase (GR)) [18]. In addition, we established also C1 decreased cortisol levels in obese Zucker rats by inhibiting the enzyme 11-beta hydroxysteroid dehydrogenase type 1 (11β-HSD1) [19].

Chemical structure of C1.

To address this gap, physicochemical property–based predictive models were employed to assess the drug potentials of C1, using the Lipinski’s “rule of five”. This widely used qualitative guideline helps determine whether compounds are likely to be effective oral drugs and suggests that a drug candidate is more likely to be poorly absorbed if it contains more than five hydrogen bond donors (nOHNH) or 10 hydrogen bond acceptors (nON) or possesses a molecular weight (MW) of greater than 500 Da, a topological polar surface area (TPSA) of greater than 140 Å, and a logarithmic partition coefficient (cLogP) value of greater than five [20]. In addition to assessed the impacts of C1 on lipid metabolism and OS in a genetic obesity model using Zucker rats to develop a safe and effective alternative obesity treatment. Additionally, as the C1 chemical structure includes carboxylic, aromatic, and thiazole functional groups, similar to those in mirabegron and other β3-ADR agonists, C1’s β3-ADR agonist activity was evaluated.

2. Materials and methods

C1 was synthesized as reported in [18], and the reference drug (CLX) was donated by MEDIX Pharmaceutical Laboratories. Glucose was measured using an Optium Xceed glucometer and FreeStyle Optium Blood Glucose Test Strips (Abbott Laboratories 04980, México). TG and FFA levels were analyzed using ultraviolet–visible (UV–vis) spectroscopy (PerkinElmer LAMBDA 25, Waltham, Massachusetts 02451, USA). Insulin levels, GSH contents, TBARS levels, and protein contents were measured using UV–vis spectroscopy (ELECTRA, Bio-Rad Laboratories, Inc., Philadelphia, Pennsylvania 19103, USA). Polymerase chain reaction (PCR) was performed using a Light-Cycler Nano Real-Time PCR System (Roche Diagnostics, Mannheim 68305, Germany).

2.1. Animals and treatments

2.1.1. Animals.

Fourteen lean male HsdHlr:Zucker–Lepr rats (LRs) and twenty-eigth obese male HsdHlr:Zucker–Leprfa/fa rats (ORs) of five-week-old were used for experiments. The animals were obtained from Harlan Laboratories (Indianapolis, IN, USA). The study design complied with the protocols established by the Norma Mexicana on the subject (NOM-062-ZOO-1999, Especificaciones Técnicas para la Producción, Cuidado y Uso de Animales de Laboratorio, SAGARPA) and was approved by the Comité Institucional de Cuidado y Uso de Animales de Laboratorio (CICUAL), ESM-CICUAL-02/20-03-2013, Escuela Superior de Medicina, IPN.

The animals were maintained at a controlled temperature (22°C ± 1°C) in a 12 h light/dark cycle (the lights were on from 7:00 p.m. until 7:00 a.m.). The rats were acclimated for 1 week before the initiation of any procedure. Food (Teklad Global Rodent Diet 2018S, Harlan®) and water were provided ad libitum.

2.1.2. Treatments.

Four control groups of Zucker rats were randomly assigned according to their body condition (lean or overweight) and the type of treatment administered (saline or a vehicle (ethanol:water, 10:90, at an ethanol dose of 0.8 mg/Kg bodyweight per day). Two groups comprised lean Zucker rats, one of which received saline (LRS, n = 7); the other, the vehicle (LRV, n = 7). The remaining two groups comprised obese Zucker rats, one of which received saline (ORS, n = 7); the other, the vehicle (obese vehicle-treated (ORV), n = 7). In addition, two experimental groups were formed: The first group included obese Zucker rats treated with CLX (ORCLX, 10 mg/Kg bodyweight per day, n = 7) and the second group received C1 (ORC1, 12 mg/Kg bodyweight per day, n = 7) dissolved in the vehicle. To enable direct comparison with CLX, C1 was administered in equimolar doses. All the treatments were intragastrically administered over 28 days because the combination of intragastric administration and the selected experimental period enabled the precise dosing of drug candidates with solid food ad libitum, facilitating the control study of human-simulated administration rates and oral dosages. In addition, these experimental conditions enabled the evaluation of metabolic and hepatic changes without causing excessive damage or irreversible effects, enabling the comprehensive assessment of metabolic alterations and the efficacy and safety of C1, as reported in other studies that using different murine models, including obese Zucker rats [21–23].

2.2. Control bodyweight, food intake, and survival percentage

The daily growth rate (DGR) and food intake (FI) of all the Zucker rats or each group were recorded using an Ohaus analytical balance (PA114, Pioneer-Series digital electronic scale) with a capacity and a sensitivity of 110 and 0.0001 g, respectively, every third day during the 28 days of treatment. The DGR was calculated using the following equation: weight gain (WG) = (final weight − initial weight)/age (days). The results were expressed in grams per unit of time (days). The FI was determined based on the weight difference—for which the amount of food was initially weighed before and after consumption (initial and final WFs, respectively)—calculated using the mathematical expression FI = initial WF (g) − final WF (g), and expressed in grams. The survival rate was calculated by dividing the number of animals that survived the treatment by the initial number of animals in each group and multiplying by 100 [24].

After 28 days of treatment according to the guidelines (NOM-062-ZOO-1999, Especificaciones Técnicas para la Producción, Cuidado y Uso de Animales de Laboratorio, SAGARPA), the animals in each group received an intraperitoneal anesthetic dose of pentobarbital (60 mg/kg). Deep anesthesia was verified based on the plantar reflex, and cardiac puncture was performed to obtain whole blood samples, it is important to mention that this was as humane endpoint for all the experimental animals. Subsequently, mesenteric and hepatic adipose tissues (ATs) were dissected, immediately frozen in liquid nitrogen, and stored at −80°C for subsequent use.

2.3. Hepatic biomarkers of antioxidant defense (GSH) and oxidative damage (LPO)

The GSH contents of all the Zucker rats were determined using Ellman’s method, because the -SH group can reduce 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB). Initially, 0.05 g of liver was homogenized with 5 mL of 3% metaphosphoric acid (MPA). Then, the sample was centrifuged at 4500 rpm for 15 min. Subsequently, 167 µL of the supernatant was diluted with 500 µL of phosphate-buffered saline (0.1 M, pH 8), 10 µL of DTNB was added, and the mixture was shaken. After 5 min, the color development was measured at 415 nm. The GSH content was determined using a GSH standard curve (from 0.0018 to 0.12 µg/mL) and expressed in micrograms of GSH per milligram of protein [18]. The protein concentration was measured at 595 nm using the Bradford method and a Cayman chemical protein determination kit (Item No. 704002, Michigan, USA), which is a colorimetric method that exploits the color change of Coomassie® dye from brown to blue when it binds to proteins in acidic media.

TBARS levels were determined using 0.1 ± 0.005 g of liver tissue homogenized in 1 mL of distilled water. Promptly, 150 µL of the homogenate was mixed with 350 µL of Tris–HCl buffer (0.15 M, pH 7.4). The mixture was incubated at 37°C for 30 min, and 1000 µL of TBA (3% w/v) dissolved in trichloroacetic acid (TCA, 15% w/v) was added. The mixture was incubated at a boil for 1 h and then centrifuged at 6000 rpm for 15 min. The supernatant’s absorbance was read at 540 nm, and the TBARS levels were determined using a 1,1,3,3-tetramethoxypropane (TMP) standard curve (16–0.25 nmol/mL) and expressed in micromoles of TBARS per milligram of protein [18]. The protein content was determined as reported for the GSH content test.

2.4. Plasmatic and blood biomarkers

2.4.1. Plasmatic insulin levels and blood glucose.

In Zucker rats, plasmatic insulin levels were determined using an ELISA-type enzyme immunoassay and the Cayman chemical insulin (rat) EIA kit (Item No. A05105, Michigan, USA). This immunoassay is based on a competitive system, where unlabeled rat insulin competes with acetylcholinesterase (AChE)-labeled insulin for a limited number of binding sites on the guinea pig anti-rat insulin antibody. The insulin-antibody complex binds to a secondary antibody (goat anti-guinea pig), immobilized on the plate. Following incubation, Ellman’s reagent is added, which acts as a chromogenic substrate for AChE. The resulting color intensity, detectable at 414 nm, is inversely proportional to the concentration of insulin in the sample. Insulin levels were expressed in ng/mL, as calculated based on a calibration curve prepared according to the manufacturer’s specifications [25].

In Zucker rats, blood glucose levels were measured using a glucometer [26]. On every third morning, blood samples were collected from the tail vein of each animal, and glucose concentrations were measured using the glucometer (Optium Xceed) and FreeStyle Optium test strips, both from Abbott. The glucose levels were recorded in milligrams per deciliter.

2.4.2. Plasmatic TG and FFA levels.

In lean and obese Zucker rats, plasmatic TG levels were measured according to the GPO–PAP method using a Randox kit (item No. TR213, West Virginia, USA). The method is based on an enzymatic reaction cascade, culminating in the formation of quinoneimine, which concentration is directly proportional to TG levels. The reaction begins with lipase-induced TG hydrolysis, releasing glycerol, which is then phosphorylated by the enzyme glycerol kinase, producing glycerol-3-phosphate, which is oxidized, generating hydrogen peroxide (H_2_O_2_). Finally, H_2_O_2_ reacts with a mixture of 4-aminoantipyrine and 4-chlorophenol, forming quinoneimine, a colored compound which absorbance is measured at 500 nm. TG levels were expressed in milligrams per milliliter [27].

In Zucker rats, plasmatic FFA levels were measured using a commercial non-esterified fatty acid kit from Randox Laboratories (item No. FA115, West Virginia, USA). The assay is based on coupled enzymatic reactions. Initially, the acylation of FFAs by acyl coenzyme synthetase produces acyl coenzyme A, which oxidizes, generating H_2_O_2_. Finally, in peroxidase, H_2_O_2_ reacts with a mixture of 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine, generating a purple adduct detectable at 550 nm. The FFA levels were expressed in mmol/L, using the expression FFA = (Pabs_sample_/Pabs_standard_)*1.04, as mentioned in the manufacturer’s instructions for the kit, where Pabs_sample_ and Pabs_standard_ are the average absorbance values of the sample and standard, respectively. The FFA levels were expressed in millimoles per liter [28].

2.4.3. Plasmatic proinflammatory biomarkers.

In Zucker rat plasma, TNF-α and IL-6 levels were evaluated using 900-K73 and 900-K86 kits (PeproTech, Ciudad de México, México), respectively. Both methods comprise a sandwich immunoassay, where the antigen is immobilized by an antibody linked to avidin peroxidase, which can generate a product detectable at 405 nm. After the construction of corresponding calibration curves, the levels of both cytokines were expressed in picograms per milliliter [29]. AT proteins (200 mg/mL) were extracted using radioimmunoprecipitation assay (RIPA) buffer (Nonidet P-40 0.625% sodium deoxycholate, 0.625% sodium phosphate (6.25 mM), and ethylenediaminetetraacetic acid (1 mM) at pH 7.4) supplemented with protease inhibitor cocktail (10 g/mL).

2.5. Real-time PCR analysis of β3-ADR and UCP-1 in mesenteric AT

Real-time quantitative (q)PCR was performed on complementary deoxyribonucleic acid (cDNA) prepared from the total ribonucleic acid (RNA) isolated from white mesenteric AT. The total cellular RNA was extracted using TRIzol^TM^ reagent. The cDNA was synthesized using a reverse transcription kit (First Strand cDNA Synthesis Transcriptor Kit, Roche Diagnostics) from total RNA samples (500 ng/µL), and the levels were normalized using 18S RNA. The real-time qPCR reaction was performed using the reverse transcription kit (First Strand cDNA Synthesis Transcriptor Kit, Roche Diagnostics). All the qPCR experiments required the standardization of the reaction efficiency to ensure accurate gene expression quantification. In this study, mRNA levels were calculated using the comparative threshold cycle method (ΔΔCt). In the standardization formula (2^−ΔΔCt^), the constant “2” represents the assumed efficiency of the PCR reaction (i.e., 100% efficiency), which is typically validated using a dynamic range curve or serial dilutions of the reference (18S housekeeping) gene. Oligonucleotide primers (Universal Probe Library, Roche) were generated using online assay design software (https://qpcr.probefinder.com/organism.jsp) and are shown in Table 1 [30].

Table 1: Primer sequences for real-time qPCR; designs are based on ensemble transcript ID of the Rat ProbeLibrary.

2.6. In silico evaluation

2.6.1. Physicochemical and toxicological properties.

The drug-likenesses of C1, CLX, and epinephrine were analyzed according to physicochemical and toxicological properties using the Molinspiration predictor (http://www.molinspiration.com/) and Osiris (https://www.organic-chemistry.org/prog/peo/), respectively. The physicochemical properties were based on Lipinski’s rule of five, while the toxicological parameters included mutagenicity, tumorigenicity, irritability, and reproductive effects [20].

2.6.2. Docking study validation.

A β3-ADR crystal (Protein Data Bank (PDB) ID: 9IJE, resolution: 2.34 Å) was used in this study; the 3D crystal structure was downloaded from the PDB (https://www.rcsb.org). Four chains comprised the receptor (A, B, C, D, N, and R) [17]. The co-crystallized ligand (epinephrine) and water molecules and other chains were removed, polar hydrogen was assumed, and partial Kollman charges were added to the β3-ADR crystal using AutoDock Tools 1.5.7 software (The Scripps Research Institute, La Jolla, CA, USA). A two-dimensional (2D) epinephrine molecule was drawn using Chemsketch. The structure was optimized using a semiempirical AM1 method and Gaussian16. Then, Gasteiger partial charges were added using AutoDock Tools.

Finally, using AutoDock Vina, epinephrine was redocked into the receptor in a 40 Å^3^ box with a 0.375 Å spacing 100 times for each compound into the redocked complex was superimposed on the co-crystallized reference complex using Visual Molecular Dynamics (VMD) 1.9.3 and the root-mean-square deviation (RMSD) was calculated using University of California at San Francisco Chimera.

2.6.3. Prediction of ligand-binding modes and affinities via molecular docking and dissociation constants (Ki).

After the molecular docking was validated, C1 and CLX were docked in the β3-ADR 3D structure, following the same procedure as that described in the validation method. Ki was calculated using the Gibbs free energy (ΔG) equation (ΔG = R T ⋅ ln Ki), where the temperature (T) is 298.15 K, the gas constant is 1.987 cal/mol·K, and the lowest ΔG value obtained using molecular modeling is used [31].

2.7. Statistical analysis

The data were expressed as means ± standard errors of means for n = 5–7 rats per group. The results were evaluated using one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. Statistically significant differences between groups were defined as p < 0.05 or p < 0.001. The data were analyzed using Prism 5.0 (GraphPad Software Inc., USA). The three variables were independently analyzed, and a Bonferroni correction was applied to control for type I error arising from multiple comparisons. Consequently, differences with adjusted p-values of less than 0.0167 and 0.0003 were considered as statistically significant, reflecting a Bonferroni correction applied to the original significance levels of 0.05 and 0.001 to control for multiple comparisons.

3. Results

3.1. Control bodyweight, FI, and survival percentage

Fig 2(a) shows no significant differences in bodyweight between the obese Zucker rats in the ORS and ORV groups. Similarly, among lean Zucker rats, LRS and LRV did not produce significant differences in bodyweight related also with the FI (Fig 2(c)). However, regardless of the administered treatment, a comparison of the lean and obese Zucker rat groups revealed a significant difference. Fig 2(b) shows that saline-treated (3.76 ± 0.60 g/day) lean Zucker rats did not show any notable DGR differences compared to those in the LRV group (4.31 ± 0.30 g/day). Similarly, among the obese Zucker rat groups, saline administration of saline (6.52 ± 0.62 g/day) or the vehicle (7.98 ± 0.77 g/day) did not appear to influence the DGR. However, the ORS and ORV groups had significantly higher DGRs than their lean counterparts (LRS and LRV, respectively), regardless of the administered treatment. Likewise, the DGRs of vehicle-treated obese Zucker rats were significantly higher than those of the rats in the ORS group. In addition, although obese ORCLX-treated (7.54 ± 0.61 g/day) and ORC1-treated (6.78 ± 0.67 g/day) Zucker rats showed lower DGRs than those in the ORV groups, the DGR of the ORC1-treated group was the same as that of the obese saline-treated group.

Zucker rat attributes evaluated under different experimental conditions.(a) Bodyweight (g) growth curves of the saline- and vehicle-treated Zucker rats in the LRS (●), LRV (o), ORS (■), and ORV (□) groups plotted as functions of age (days), (b) average DRG rates of the C1, CLX, and vehicle-treated lean (LR) and obese (OR) rats, (c) cumulative food consumption of the rats in the LRS (●), ORV (□), ORCLX (▲), and ORC1 (♦) groups, and (d) survival rates (percentages) of the differently treated LR and OR. The lines represent the observed survival rates; the y-axis represents the survival proportion, and the x-axis represents the treatment time (28 days). ap < 0.0001 against the LRS group, bp < 0.0001 compared with the LRV group, cp < 0.0001 vs. the ORS group, and dp < 0.0001 relative to the ORV group. A comparison of the survival curves shows no significant differences were obtained over time using the Kaplan–Meier statistic and log-rank (Mantel–Cox) test.

Notably, Zucker rats in the LRS, LRV, ORS, and ORC1 groups showed 100% survival rates. However, obese Zucker rats vehicle (28.6%) and CLX (14.3%) recorded mortalities during treatment, with the loss of two individuals in the ORV group and one in the ORCLX group, as shown in Fig 2(d).

3.2. Hepatic biomarkers of antioxidant defense (GSH) and oxidative damage (LPO)

Fig 3(a) shows that the hepatic GSH levels of the obese Zucker rats in the ORS (3.67 ± 1.20 µg/mg protein) and ORV (3.12 ± 0.67 µg/mg protein) groups remained practically constant. However, these GSH levels were lower than those of the lean rats in the LRS (5.28 ± 0.99 µg/mg protein) and LRV (4.97 ± 1.26 µg/mg protein) groups. Furthermore, compared to the hepatic GSH levels of the control groups, those of the obese CLX-treated Zucker rats decreased, while those of both lean rat (LRS and LRV) groups were not restored. In contrast, among all the groups, the hepatic GSH levels of the obese C1-treated Zucker rats (9.05 ± 2.08 µg/mg protein) increased, reaching statistical significance against the those of the group of ORV.

Antioxidant and oxidant hepatic markers of Zucker rats under different treatments.(a) Average GSH and (b) TBARS levels. The numbers of samples in each group were LRS = 7, LRV = 7, ORS = 7, ORV = 5, ORCLX = 6, and ORC1 = 7. Each experiment was performed in triplicate; ap = 0.0245 against the OVR group.

Fig 3(b) shows that although the TBARS levels of the lean saline- (611.17 ± 86.72 nmol/mg protein) and vehicle-treated (604.59 ± 199.27 nmol/mg protein) Zucker rats were similar, they were lower than those of the obese rats in the ORS (908.89 ± 164.40 nmol/mg protein) and ORV (847.73 ± 165.82 nmol/mg protein) groups. Likewise, Fig 3(b) shows that the TBARS levels of the CLX-treated rats decreased by approximately one-half compared to those of both obese rat groups. In contrast, the TBARS levels of the C1-treated animals increased (315.71 ± 144.99 and 609.57 ± 119.58 nmol/mg protein) compared to those of the ORCLX group.

3.3. Plasmatic and blood biomarkers

3.3.1. Blood glucose and plasmatic insulin levels.

The blood glucose levels of the ORV (97 ± 4 mg/dL) and LRV (90 ± 2 mg/dL) groups were similar (Fig 4(a)). The blood glucose level (103 ± 4 mg/dL) of the ORCLX group was significantly different (p = 0.0020) compared to that of the LRV group. A similar effect was observed after C1 administration (112 ± 4 mg/dL). Despite these between-group differences, no animal developed hyperglycemia or T2DM because all the blood glucose levels were below 140 mg/mL (Fig 4(a)), as reported in previous studies [32,33]. Fig 4(b) shows that the Zucker rats in the LRS and LRV groups had plasmatic insulin levels of 0.60 ± 0.25 and 0.38 ± 0.12 mg/mL, respectively. However, the plasmatic insulin levels of the ORS, ORV, ORCLX, and ORC1 groups were 2.56 ± 0.68, 2.36 ± 0.65, 2.45 ± 0.97, and 1.51 ± 0.44 mg/mL, respectively.

Blood glucose and insulin levels of Zucker rats after 28 days of different treatments.(a) Average blood glucose and (b) plasmatic insulin levels of control and experimental groups. The numbers of samples in each group was: LRS = 7, LRV = 7, ORS = 7, ORV = 5, ORCLX = 6, and ORC1 = 7. Each experiment was performed in triplicate; ap = 0.0002 against LRS and bp = 0.0002.

3.3.2. Plasmatic TG and FFA levels.

Fig 5(a) shows that the plasma TG levels of the lean saline- (47.45 ± 3.94 mg/dL) and vehicle-treated (29.50 ± 3.86 mg/dL) rats were similar and lower than those of the obese Zucker rats. In addition, the obese saline- (374.13 ± 29.91 mg/dL), vehicle- (344.01 ± 53.71 mg/dL), and CLX-treated (461.09 ± 83.95 mg/dL) rats presented similar plasma TG levels. In contrast, obese C1-treated Zucker rats showed significantly higher TG levels (811.77 ± 58.13 mg/dL). Fig 5(b) shows that the FFA levels of the lean saline- (0.39 ± 0.10 nmol/L) and vehicle-treated (0.39 ± 0.03 nmol/L) rats were lower than those of the obese Zucker rats. However, the FFA levels of the obese CLX-treated (0.72 ± 0.17 nmol/L) rats were lower than those of the saline- (1.15 ± 0.22 nmol/L), vehicle- (1.09 ± 0.07 nmol/L), and C1-treated (0.91 ± 0.07 nmol/L) rats.

Correlations among the plasmatic lipid profiles of Zucker rats after 28 days of different treatments.(a) TG and (b) FFA levels. The numbers of samples in each group were LRS = 7, LRV = 7, ORS = 7, ORV = 5, ORCLX = 6, and ORC1 = 7. Each experiment was performed in triplicate; ap < 0.0001 against LRS, bp < 0.0001 compared to LRV, cp < 0.0001 compared to ORS, dp < 0.0001 against ORV, and ep = 0.0002 against ORCLX.

Because no changes in the lipid profiles and antioxidant/oxidant markers were observed in the lean LRS and LRV neither in obese Zucker rats ORS and ORV, only the plasmatic TNF-α, IL-6, β3-ADR, and UCP-1 levels were determined in the LRV, ORV, ORCLX, and ORC1 groups because they shared the same environment.

3.3.3. Plasmatic proinflammatory biomarkers.

Fig 6(a) shows that the plasmatic TNF-α levels significantly increased from 18.4 ± 2.2 to 35.3 ± 2.4 pg/mL between the LRV and ORV groups. In addition, in the ORCLX group, the TNF-α level (52.3 ± 5.7 pg/mL) was significantly higher than those in the ORC1 (18.4 ± 2.2 pg/mL) and ORV groups. Fig 6(b) shows that compared with the plasmatic IL-6 level of the lean animals (249 ± 13 pg/mL), IL-6 level of the ORV group considerably increased, while those of the CLX (177 ± 26 pg/mL) and C1-treated (166 ± 13 pg/mL) groups decreased, although these differences were not significant.

Plasmatic cytokine levels of Zucker rats under different treatments.(a) TNF-α and (b) IL-6 levels. The numbers of samples in each group were LRV = 7, ORV = 5, ORCLX = 6, and ORC1 = 7. Each experiment was performed in triplicate; a,b,cp = 0.0073 against ORV, LRV, and ORCLX, respectively.

3.4. Real-time PCR analysis of β3-ADR and UCP-1 in mesenteric AT

In the LRV and ORV groups, CLX had no effect on the gene expressions (messenger (m)RNAs) of β3-ADR and UCP-1 (Fig 7(a) and 7(b), respectively) in mesenteric AT. In contrast, in the ORC1 group, the expressions of both genes in mesenteric AT significantly increased in response to C1.

Relative mRNA expression levels.(a) β3-ADR and (b) and UCP-1 in the AT of Zucker rats under different treatments. The numbers of samples in each group were LRV = 7, ORV = 5, ORCLX = 6, and ORC1 = 7. Each experiment was performed in triplicate; ap = 0.0149 and bp = 0.0090 compared with LRV, ORV, and ORCLX.

3.5. In silico evaluation

3.5.1. Physicochemical and toxicological properties.

The drug-likeness properties determined using Lipinski’s rule of five are shown in Table 2. The MWs of epinephrine, C1, and CLX were within the recommended range of 500 Da. In addition, the TPSAs, hydrogen bond acceptors (nON), and hydrogen bond donors (nOHNH) of these three compounds agreed. Among the three compounds, only epinephrine did not satisfy the miLogP criterion, indicating that epinephrine was more hydrophilic than C1 and CLX [20,34].

Table 2: Physicochemical and toxicological properties of the compounds.

Furthermore, compared with C1 and CLX, epinephrine had lower MW, nON, and nOHNH values. However, the TPSA of epinephrine was smaller than that of C1 and larger than that of CLX. In addition, C1 did not pose any mutagenic, tumorigenic, irritant, or reproductive risks, similar to the safety profile of epinephrine.

3.5.2. Docking method validation.

Fig 8(a) shows the overlaid images of the redocked and native epinephrine structures in the β3-ADR crystal. The protocol implemented using AutoDock Vina correctly reproduced the crystal conformation (Fig 8(b)), with a RMSD of 2.6 Å with respect to the redocked epinephrine structure (Fig 8(c)). As reported in previous studies, hydrogen bonds were the key interactions among the SER208, SER212, ASN117, and ASN332 moieties and the β-OH, catechol, and alkylamine groups in epinephrine. The crystallized and redocked epinephrine structures interacted with β3-ADR at distances longer than 5.0 Å, with a ∆G value of −6.3 kcal/mol and an inhibition constant (Ki) of 24.07 µM, indicating correct interactions and orientations at the active site and confirming that the protocol adequately reproduced both the position and critical interactions of the ligand, validating the protocol’s applicability to evaluate the CLX and C1 binding modes.

Validation of the β3-ADR–epinephrine complex docking.(a) Superposition of the co-crystallized (pink) and redocked (green) complexes at the β3-ADR active site. (b) A 3D representation of epinephrine co-crystalized at the orthosteric site. (c) Epinephrine redocked using VMD at the receptor’s active site.

According to multiple studies, RMSDs of < 2Å are convenient parameters to generate a trustworthy model [35]. However, previous studies have also shown that RMSDs can be modified depending on the ligand characteristics. Reportedly, although rotatable bonds substantially increase the RMSD, the ligand is not necessarily at the binding site [36]. Specifically, epinephrine features one rotatable bond within an alkylamine group. Another factor that may affect the RMSD is the resolution of the crystal structure, as a resolution of approximately 1 Å is considered as optimal for docking studies. Because the resolution of the 9IJE protein is above 2.0 Å, this can impact the accuracy of ligand–receptor recognition because of the limitations of the crystal structure. Therefore, the results revealed that the model used in the docking study is useful to predict C1 and CLX interactions because the redocked ligand was embedded at the active site of the receptor via anchoring distances and interactions.

3.5.3. Prediction of ligand-binding modes and affinities using the molecular docking inhibition constant (Ki).

The bindings of C1 and CLX at the β3-ADR active site are shown in Fig 9(a). The active residues involved in forming the β3-ADR–ligand complex were PHE308, PHE309, ASN332, ASN3121, ASP117, VAL121, SER208, and SER 212. In the β3-ADR–CLX complex, the involved amino acid residues established hydrogen bonds between ASP117 (4.72 Å) and the CLX amino group, as well as π–alkyl interactions between THR122 (4.93 Å) and LEU329 (6.00 Å) residues and aromatic rings and a π–π interaction between the ligand’s amino group and the PHE309 residue (4.47 Å) (Fig 9(b)). By contrast, in the β3-ADR–C1 complex, conventional hydrogen bonds formed among C1 hydroxyl and secondary amine groups and ASP117 (5.27 Å), VAL121 (2.82 Å), and THR122 (2.55 Å) residues as well as π–π and π–alkyl interactions between both PHE309 (4.47 Å) and LEU309 (329 Å) residues and the 5-aminosalicylic acid (5-ASA) ring (Fig 9(c)). In addition, a π–σ interaction formed between VAL118 (4.20 Å) and the six-membered ring of 5-ASA. Furthermore, C1 (ΔG = −9.1 kcal/mol, Ki = 1.15 µM) showed a higher binding affinity than CLX (ΔG = −8.8 kcal/mol, Ki = 4.45 µM) to the β3-ADR active site, as evidenced by its more negative free binding energy.

Representation of ligands docking at the β3-ADR active site.(a) Three-dimensional representations of the co-crystallized ligand epinephrine (pink), C1 (purple), and CLX (yellow) in the binding cleft of the adrenoreceptor (PDB ID: 9IJE) and 3D representations of (b) CLX and (c) C1 docked at the active site of the receptor, as modeled using VMD.

4. Discussion

The genesis of obesity is the chronic and excessive accumulation of body fat because of a sustained energy imbalance between caloric intake and energy expenditure. Multiple factors, both environmental and genetic, converge in the etiology of obesity. Among the environmental factors, eating habits, sedentary lifestyles, and a total lack of physical activity stand out. In contrast, genetic predisposition includes various biological factors that favor excess lipid storage in AT, with genetic alterations being a key component in the pathogenesis of obesity [37]. In this context, animal models represent an essential strategy for investigating pathophysiological mechanisms involved in the development and progression of obesity and evaluating possible therapeutic interventions [38]. Obese Zucker rats are the best-established animal model of genetic obesity and in vivo testing. This model, including obese and lean Zucker rats, represents two MS extremes and provides valuable information on various human metabolic disorders. In Zucker fa/fa rats, obesity is caused by a mutation in the leptin receptor, leading to hyperphagia and the early onset of NAFLD between 3 and 5 weeks of age. In contrast, lean Zucker rats have normal metabolic function and are ideal controls for comparative studies [39,40].

In this context, this study found that both groups of obese Zucker rats, one treated with a saline solution and the other with a vehicle for 28 days, showed approximately doubled increases in weight, DGR, and FI compared with those of the counterpart groups of nonobese Zucker rats, which received either the saline solution or vehicle, respectively. This difference suggests that eight-week-old obese Zucker rats exhibit polyphagia and abnormalities in lipid metabolism, which contribute to obesity, as described in previous studies [41,42].

Additionally, compared to lean rats, obese Zucker rats had lower hepatic GSH levels, regardless of the treatment (saline solution or vehicle), suggesting that obese rats had lower methionine levels, limiting the GSH antioxidant synthesis rate [39]. This could be related to an imbalance in cellular defense mechanisms against ROSs [43]. In contrast, lean rats showed higher GSH levels, indicating higher antioxidant capacity. These results suggest that obesity is associated with an altered redox state [44], which could explain the increased TBARS levels in the livers of the obese rats, driving the development of NAFLD by damaging mitochondria and cell membranes via LPO, as previously described [45].

Likewise, plasmatic TG, FFA, glucose, and insulin levels were simultaneously increased, suggesting that a metabolic alteration characteristic of insulin resistance (IR) is typical for these Zucker rats. Therefore, these results support the coexistence of dyslipidemia, hyperglycemia, hyperinsulinemia, and OS in the context of obesity, as reported in previous studies [40,46].

However, herein, no differences in hepatic and plasmatic parameters were noted between the obese and counterpart lean Zucker rats treated with the vehicle and those treated with saline. This absence of changes within each body condition (obese or lean) group suggests that alcohol (0.8-mg/kg bodyweight) intragastrically administered to obese and lean Zucker rats did not cause any adverse or toxic effects, as the alcohol’s concentration was well below the toxicity threshold (2500-mg/kg bodyweight) [47]. However, a mortality rate of 28.6% was reported in obese Zucker rats, possibly because chronic alcohol administration contributes to the development of cardiovascular disorders in obese rats, as previously reported in studies on animals and humans [48,49], even if the rats did not have any previous liver damage.

Therefore, the Zucker rat obesity-plus-alcohol-intake model used in this study is an appropriate tool for evaluating the antiobesity and antioxidant effects of C1, a synthetic compound containing phenol, salicylate, and benzothiazole residues in its chemical structure, using CLX (Asenlix®, Aventis) as the reference drug, as CLX has previously been described as a strategy for treating obesity in Latin American countries, because, as an amphetamine derivative, CLX increases the release of neurotransmitters, such as norepinephrine and dopamine, in the central nervous system. These neurotransmitters stimulate α- and β-adrenergic receptors, which induce weight loss through an anorexic effect [13,50].

These results indicated that DGR and FI values significantly decreased in obese Zucker rats treated with CLX compared to those treated with the vehicle alone. These decreases could be attributed to the psychostimulant activity of CLX, which suppressed appetite and increased energy expenditure in obese Zucker rats [51,52], promoting weight loss, as observed in other murine models where the effects of amphetamine derivatives on weight loss were evaluated [53]. By contrast, the DGR values of obese C1-treated Zucker rats were lower than those of obese vehicle- and CLX-treated Zucker rats, suggesting that C1 significantly reduced both adipocyte size and lipid droplet number in a manner similar to that of thioflavin T, as both compounds share a benzothiazole group [54]. Furthermore, C1 could promote thermogenesis, which would increase energy expenditure and concomitant weight loss in obese Zucker rats, even under the condition of a higher FI, as previously reported for other salicylate derivatives [55,56].

Additionally, our study revealed that CLX significantly decreased GSH levels in obese rats compared to those in the LRS, LRV, ORS, ORV, and ORC1 groups because when amphetamines are metabolized by the CYP2D6 enzyme, a toxic epoxide is generated and forms covalent adducts with GSH, reducing its availability. Likewise, after CLX administration, released dopamine can autooxidize via the Fenton reaction, generating highly reactive free radicals and nitrogen species, such as nitric oxide (NO) and peroxynitrite (ONOO^−^), contributing to mitochondrial dysfunction and further depleting GSH, thereby exacerbating hepatic OS in obese rats. Paradoxically, CLX reversed LPO, possibly because of the induction of antioxidant enzymes, as amphetamine derivatives reportedly induce catalase expression, which attenuates lipid damage [57–61]. However, the absence of LPO does not rule out other forms of cellular toxicity, including protein alterations, mitochondrial damage, and neurotransmission dysfunctions, which explain the death of 14.3% of the CLX-treated animals.

Several studies have shown that the administration of antioxidants, through compounds that either reduce ROS levels or restore GSH concentrations, can prevent or mitigate obesity-induced toxic effects. In animal models, flavonoids (polyphenolic compounds) and sulfur compounds induce the expression of the enzyme glutamate–cysteine ligase (GCL), which catalyzes the rate-limiting step in cytosolic GSH biosynthesis via a finely regulated process [62,63]. In this context, because of its good antioxidant and hepatoprotective properties, C1 was administered to obese Zucker rats. Previous studies have shown that C1 exhibits remarkable antiradical activity against hydroxyl radicals (^•^OH) generated during the Fenton reaction and protects against acetaminophen-overdose-induced liver damage in CD-1 mice [18].

In this study, obese C1-treated Zucker rats showed significantly increased hepatic GSH levels, related to the sulfur atoms and phenolic group in C1’s chemical structure, which can stimulate endogenous GSH synthesis. Simultaneously, C1 protected against lipid damage, as it decreases TBARS levels. This protective effect can be attributed to C1’s ability to inhibit ^•^OH radical formation, thus preventing the cleavage of the hydrogen atom from the methylene group (–CH_2_–) in lipids [64], as previously described for other polyphenolic compounds [65,66]. These findings suggest that C1 exerts protective effects against obesity-associated NAFLD in Zucker rats, as 100% survival was also reported for the groups of lean rats (LRS and LRV) groups, which did not show any signs of liver damage.

Epidemiological studies have identified NAFLD as the hepatic component of nonalcoholic steatohepatitis (NASH) and have found that IR represents its distinctive pathophysiological hallmark [67]. According to the results of this study, compared to lean vehicle-treated rats, obese vehicle- and CLX-treated Zucker rats showed significantly higher plasmatic glucose and insulin levels because of the decreased ability of peripheral tissues to adequately respond to insulin in obese rats, leading to inefficient glucose uptake and, consequently, hyperglycemia [67]. In response to this decreased insulin sensitivity, pancreatic beta cells increase insulin secretion as a compensatory mechanism, generating hyperinsulinemia [68]. These findings are consistent with those reported in other studies, indicating that these conditions are characteristic of obese Zucker rats [40].

In contrast, obese C1-treated Zucker rats had significantly higher blood glucose levels compared to those of the control and experimental groups but showed lower circulating insulin levels than the ORV and ORCLX groups. This response suggests that under C1 treatment, insulin sensitivity has not yet been fully restored, possibly because of progressive pancreatic beta cell failure. This phenotype supports the hypothesis that chronic hyperglycemia imposes a sustained burden on beta cells, exceeding their functional capacity and compromising the maintenance of normoglycemia [68,69]. Thus, although C1 may exert a partially protective effect on IR, this effect is limited in the context of impaired pancreatic beta cell function.

In addition to presenting hyperinsulinemia and hyperglycemia, obese Zucker rats exhibit elevated circulating levels of TG and cholesterol, typical manifestations of MS [70], findings that are consistent with the results of this study, where obese Zucker rats in the ORV group showed significantly higher plasmatic TG concentrations compared to those of the rats in the LRV group. This dyslipidemia is associated with a mutation in the leptin receptor (fa/fa) and hyperphagia, regulated in part by altered activity in the medial prefrontal cortex. In addition, obese animals had significantly higher FFA levels than their lean counterparts, suggesting altered lipolytic turnover. This imbalance favors a lipotoxic environment [71], which is supported by increased LPO products, such as TBARS, classic indicators of lipid damage.

Likewise, this study revealed that obese Zucker rats treated with vehicle-dissolved CLX and C1 had significantly higher TG levels compared to those of the obese control group, suggesting possible underlying liver damage. However, both treatment groups showed significantly lower plasmatic FFA levels, indicating reduced lipotoxicity, consistent with the observed decreased TBARS and increased GSH levels. These findings reinforce the hypothesis that TG accumulation does not necessarily reflect a pathological state but may act as a protective mechanism by sequestering cytotoxic FFA and, thus, reducing OS [72,73]. However, these animals can suffer from cardiovascular diseases typical of T2DM [74], which require further research to confirm or rule out such pathologies.

However, several studies have reported that elevated TG levels are associated with chronic inflammation, which is characterized by increased TNF-α and IL-6 levels, which alter insulin signaling and lipid metabolism, promoting IR and perpetuating dyslipidemia [75]. In this context, compared to the ORV group, C1 decreased IL-6 levels and maintained constant plasmatic TNF-α levels, suggesting that although C1 modulates the production and activity of these cytokines, its effects are not potent enough to alter TNF-α levels to the basal state compared to that in lean Zucker rats, possibly because more TA must be eliminated before proper adipocyte function is restored to observe changes in plasmatic TNF-α levels, as demonstrated in studies on humans treated with a three-week low-calorie diet and balneotherapy [76]. These findings suggest that C1 indirectly protects against inflammation by inhibiting TNF-α-mediated ROS generation, as has previously been demonstrated using other polyphenolic antioxidant systems [77], or possibly by inhibiting IL-6 expression, as has previously been described for benzothiazole derivatives [78].

By contrast, the administration of CLX to obese Zucker rats increased TNF-α and IL-6 levels compared with those in the LRV and ORC1 groups because of the increased DNA-binding activity of activator protein 1 (AP-1), a transcription factor that induces the expressions of inflammatory genes encoding TNF-α and IL-6 genes, mediated by the underlying OS stimulus associated with CYP2D6-induced CLX metabolism, similar to that reported in C57BL/6 mice treated with an intraperitoneal dose of methamphetamine (10 mg/kg bodyweight) [79].

Likewise, C1 acted as a β3-ADR agonist, as it positively stimulated the expression of this receptor, and browned white AT in a manner similar to those of endogenous catecholamines, antidiabetics (metformin), antioxidants (vitamin E and polyphenols), or agonists, such as SR 58611, BRL 37344, CGP 12177, mirabegron, solabegron, BRL 27344, and ritobegron [16,17,80], which stimulate the expressions of thermogenic genes, such as UCP-1, in white AT [81–83]. Therefore, C1 promotes the uncoupling of the H^+^ gradient, which, in turn, favors the release of energy as a heat gradient [15], contributing to weight loss in obese Zucker rats.

By contrast, the administration of CLX to obese rats did not induce the activations of β3-ADR and UCP-1 genes. This lack of response is attributed, in part, to CLX failing to reduce chronic AT inflammation, characterized by persistently elevated TNF-α and IL-6 levels. These proinflammatory cytokines negatively interfere with β3-ADR expression, thereby inhibiting the activation of BAT-dependent thermogenic pathways. Consequently, in CLX-treated animals, weight loss appears to be mediated by increased peripheral lipolysis [84] rather than the direct stimulation of thermogenesis.

Importantly, in obese Zucker rats, the C1- and CLX-induced activation, or lack thereof, of β3-ADR is attributed to their chemical characteristics. Reportedly, most β3-ADR-related ligands share two structural domains: an aryl ethanolamine or an aryloxypropanolamine group and polar groups linked via aromatic and/or aliphatic fractions [85]. In this regard, C1 has the following structural characteristics: the arylethanolamine group is provided by the 5-ASA fragment, the aromatic rings are derived from benzothiazole, and the aliphatic fraction acts as a linking group between both fractions.

However, CLX only has an aromatic ring and an aliphatic group, reducing its affinity for β3-ADR compared to that of C1, as CLX has fewer interactions with key residues (such as ASP117, SER169, SER209, SER212, and PHE309) at the receptor binding site [16,17]. According to in silico models, in the PDB:9IJE crystal structure, C1 binds to the orthosteric site of β3-ADR via hydrogen bonds with VAL121, THR122, and ASP117 at distances of between 5.27 and 2.25 Å, improving the ΔG value. In addition, C1 establishes other π–π, π–σ, and π–alkyl interactions with PHE309, VAL118, and LEU329, respectively, at distances shorter than 4.5 Å, decreasing ligand–receptor dissociation. However, as CLX only interacts via hydrogen bonds, contributing to higher bond energies and Ki values, C1 exerted an agonistic effect on β3-ADR, which is comparable to that of mirabegron [86], solabegron, isoproterenol [87] and carazolol [17], as it interacts with ASP117, a very important amino acid in recognizing the R chain in β3-ADR. In addition, C1 presents partition coefficients similar to those of CLX, meaning that C1 is absorbed correctly and has more hydrogen acceptor and donor bonds, enabling it to interact more strongly with β3-ADR, as indicated by molecular modeling. Similarly, in silico modeling revealed that compared with CLX, C1 does not pose any health risks and is therefore a promising alternative for treating obesity and hypercortisolism-related diseases, as C1 decreases cortisol levels by inhibiting enzyme 11-βHSD1 [20].

5. Conclusion

We have demonstrated that chronic C1 administration produces daily growth loss in obese Zucker rats and is associated with the activations of β3-ADR and UCP-1 and increased lipolysis, suggesting that C1 exerts incipient systemic anti-inflammatory effects on TNF-α and IL-6 as well as locally and systemically protects against OS because C1 participates in the hepatic synthesis of endogenous antioxidant systems. Therefore, C1 can be a suitable candidate for developing therapies against obesity and other metabolic disorders.