The Allium Derivate Propyl Propane Thiosulfinate Exerts Anti-Obesogenic Effects in a Murine Model of Diet-Induced Obesity

  Citation: Liébana-García, R.; Olivares, M.; Rodríguez-Ruano, S.M.; Tolosa-Enguís, V.; Chulia, I.; Gil-Martínez, L.; Guillamón, E.; Baños, A.; Sanz, Y. The Allium Derivate Propyl Propane Thiosulfinate Exerts Anti-Obesogenic Effects in a Murine Model of Diet-Induced Obesity. Nutrients 2022, 14, 440. https://doi.org/10.3390/ nu14030440 Academic Editors: Miguel Romero-Pérez and Juan Manuel Duarte Pérez Received: 20 December 2021 Accepted: 14 January 2022 Published: 19 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). nutrients Article The Allium Derivate Propyl Propane Thiosulfinate Exerts Anti-Obesogenic Effects in a Murine Model of Diet-Induced Obesity Rebeca Liébana-García1, Marta Olivares 1,* , Sonia M. Rodríguez-Ruano 2, Verónica Tolosa-Enguís1, Isabel Chulia 1, Lidia Gil-Martínez 3, Enrique Guillamón3, Alberto Baños 3and Yolanda Sanz 1 1Institute of Agrochemistry and Food Technology, Spanish National Research Council (IATA-CSIC), 46980 Valencia, Spain; [email protected] (R.L.-G.); [email protected] (V.T.-E.); [email protected].es (I.C.); [email protected] (Y.S.) 2Department of Microbiology, Faculty of Sciences, University of Granada, 18011 Granada, Spain; [email protected] 3DMC Research Center, 18620 Granada, Spain; [email protected] (L.G.-M.); [email protected] (E.G.); [email protected] (A.B.) *Correspondence: m.olivar[email protected]; Tel.: +34-963900022 Abstract: Allium species and their organosulfur-derived compounds could prevent obesity and metabolic dysfunction, as they exhibit immunomodulatory and antimicrobial properties. Here, we report the anti-obesogenic potential and dose-dependent effects (0.1 or 1 mg/kg/day) of propyl propane thiosulfinate (PTS) in a murine model of diet-induced obesity. The obesogenic diet increased body weight gain and adipocyte size, and boosted inflammatory marker (Cd11c) expression in the adipose tissue. Conversely, PTS prevented these effects in a dose-dependent manner. Moreover, the higher dose of PTS improved glucose and hepatic homeostasis, modulated lipid metabolism, and raised markers of the thermogenic capacity of brown adipose tissue. In the colon, the obesogenic diet reduced IL-22 levels and increased gut barrier function markers (Cldn3, Muc2, Reg3g, DefaA); however, the highest PTS dose normalized all of these markers to the levels of mice fed a standard diet. Gut microbiota analyses revealed no differences in diversity indexes and only minor taxonomic changes, such as an increase in butyrate producers, Intestimonas and Alistipes, and a decrease in Bifidobacterium in mice receiving the highest PTS dose. In summary, our study provides preclinical evidence for the protective effects of PTS against obesity, which if confirmed in humans, might provide a novel plant-based dietary product to counteract this condition. Keywords: obesity; microbiota; propyl propane thiosulfinate; Allium; dose effect 1. Introduction Obesity is characterized by excessive body fat accumulation, which increases the risk of suffering from chronic diseases such as heart failure, cancer, and type 2 diabetes [ 1 ]. Many single-nucleotide polymorphisms are associated with the development of obesity; however, these differences in the genetic background alone do not explain the incidence of obesity [ 2 , 3 ]. Indeed, the constant rise in obesity prevalence can only be explained by broad environmental changes, given the stasis of human biology within the same time frame [ 4 ]. Changes in food systems and dietary patterns undoubtedly influenced the epidemiology of obesity. In this respect, hypercaloric diets disrupt the energy balance, immune system, and gut microbiome, whereas a balanced, plant-based diet protects from fat deposition and metabolic impairment [5]. Some of the health benefits associated with the intake of vegetables are due to their content in bioactive compounds. Accordingly, the administration of extracts rich in certain bioactive compounds to obese human subjects [ 6 – 8 ] or rodents [ 9 , 10 ] is proven to Nutrients 2022,14, 440. https://doi.org/10.3390/nu14030440 https://www.mdpi.com/journal/nutrients Nutrients 2022,14, 440 2 of 15 reduce some of the features associated with obesity and its comorbidities (e.g., decreases in body weight and plasma cholesterol levels or improvements in waist/hip and LDL/HDL ratios). Among the bioactive compounds currently stirring interest are the organosulfur compounds obtained from vegetables of the genus Allium—such as onions, garlic, or leeks— synthesized by these plants as a defense mechanism against tissue damage. The fact that these organosulfur compounds have functional properties exerting antibacterial, antifungal, anti-inflammatory, or antioxidant activities might underlie the century-old folk wisdom that vouches for their beneficial properties [11,12]. The most widely studied organosulfur compound is the so-called allicin (diallyl thiosulfinate). In the context of obesity, allicin is shown to curb obesity in animal models, an effect that could be, at least partly, mediated by the gut microbiota [ 13 , 14 ]. However, one limitation of allicin is that it is precariously unstable, and its bioavailability raises doubts [ 15 ]. To a lesser extent, other organosulfur compounds from the Allium genus were also investigated, such as propyl-propane thiosulfonate (PTSO) or propyl propane thiosulfinate (PTS). In this respect, the compound PTSO displayed beneficial effects on two murine models of colitis—induced by the dextran sodium sulfate (DSS) and the 2,4dinitrobenzene sulfonic acid (DNBS)—by reducing different pro-inflammatory mediators and improving the integrity of the intestinal epithelial barrier [ 16 ]. These two mechanisms are also pinpointed by Vezza et al. to explain the anti-obesogenic effects of this compound in a very recent in vivo study [ 17 ]. Regarding PTS, there is evidence of its potential use as an additive for animal nutrition [ 18 ] and its antimicrobial and anti-coccidial properties in vitro [ 19 , 20 ]. Moreover, PTS was demonstrated to be toxicologically safe in rats [ 21 ]. However, to our knowledge, research so far has not investigated PTS in the context of its potential protective effects on metabolic health. In the present study, we describe the anti-obesogenic potential of the Allium derivate PTS, tested at two different doses in a murine model of diet-induced obesity. We describe how PTS administration prevents body weight gain and the metabolic impairment associated with a hypercaloric diet in a dose-dependent manner. We also shed light on its mode of action through the effects exerted on immune mediators and energy homeostasis. 2. Materials and Methods 2.1. Mice and Treatments The experiments were performed with a total of 32 C57BL/6J mice (7-week-old males, Charles River Laboratories, Écully, France). Mice were housed in groups of four per individually ventilated cage under a 12 h light/dark cycle (lights on at 8:00) in a temperaturecontrolled room (23 ± 2 ◦ C). Mice were acclimatized for 10 days with a standard diet. During the experiment, mice had ad libitum access to water and food. The experiment was approved and performed following European Union 2010/63/UE and Spanish RD53/201 guidelines, approved by the ethics committee of the University of Valencia (Animal Production Section, SCSIE, University of Valencia) and authorized by the competent authority (Generalitat Valenciana). Furthermore, all efforts were made to guarantee the three Rs before performing the experiment. Accordingly, we considered alternatives to animal experimentation (replacement), but given its unavoidability we paid attention to reducing the number (reduction) and minimizing the suffering of, or harmful effects (refinement) on, the experimental animals. The Generalitat Valenciana authorized the procedure under the title “Evaluation of bioactive compounds obtained from plant sources” and assigned the code 2020/VSC/PEA/0088 on 26 May 2020. Randomization of mice into four experimental groups (n= 8) was conducted based on body weight to minimize baseline differences. For 10 weeks, mice were treated with: (1) control diet (D12450K Ssniff; 10% of energy from fat and no sucrose), (2) high-fat high-sugar diet (HFHSD, D12451 Ssniff; 45% of energy from lard, and 35% from sucrose), (3) HFHSD plus low-dose PTS (0.1 mg/kg/day), and (4) HFHSD plus high-dose PTS (1 mg/kg/day). Table S1 shows the detailed composition of the diets. PTS from Allium cepa L. (95% purity) was developed and provided by DOMCA SAU (Granada, Spain). The Nutrients 2022,14, 440 3 of 15 compound was administrated daily by oral gavage, whereas the control groups received PBS. Trends in body weight and food and water intake were monitored twice per week. In the 10th week, animals were fasted, anesthetized with isoflurane, and sacrificed by cervical dislocation. Blood samples were collected in EDTA tubes, and plasma was immediately transferred after centrifugation (12,000 × g, 3 min). One aliquot of plasma was kept on ice to assess intestinal permeability, and one aliquot was stored at − 80 ◦ C for biochemical and immune analyses. A section of white adipose tissue (WAT) was immersed in a solution of 4%paraformaldehyde for histological analysis. Liver, brown adipose tissue (BAT), WAT (subcutaneous (inguinal) and visceral (epididymal)), two sections of the intestine (ileum and colon), and the cecal content were snap-frozen in liquid nitrogen and stored at −80 ◦C until use. 2.2. Oral Glucose Tolerance Test An oral glucose tolerance test (OGTT) was conducted at week 9 after 4 h fasting. Blood from the saphenous vein was collected at 0, 15, 30, 60, and 120 min after an oral glucose challenge (2 g/Kg). Glucose was measured using glucose test strips (CONTOUR ® -Next meter, Bayer, Leverkusen, Germany). 2.3. Intestinal Permeability Fluorescein isothicyanine (FITC)-dextran 4 kDa (Sigma-Aldrich, St. Louis, MO, USA, 600 mg/Kg) was administrated by oral gavage one hour before the sacrifice. Plasma was diluted in an equal volume of PBS, and fluorescence was measured at the excitation wavelength of 485 nm and 535 nm emissions (CLARIOstar ® Plus Multi-Mode Microplate Reader, Ortenberg, Germany). Standard curves were obtained by diluting FITC-dextran in the plasma of non-treated mice, as previously described [22]. 2.4. Biochemical Analyses Plasma levels of insulin, glucose, triglycerides, cholesterol, and non-esterified fatty acid (NEFA) were measured with the following commercial kits: ultrasensitive insulin ELISA kit (Mercodia, Uppsala, Sweden), glucose liquid (Química Analítica Aplicada SA, Tarragona, Spain), triglyceride colorimetric assay kit (Elabscience, Houston, TX, USA), cholesterol liquid kit (Química Analítica Aplicada SA, Spain), and NEFA colorimetric assay kit (Elabscience, USA), respectively. The HOMA-IR index was calculated as fasting plasma insulin (mU/L) ×fasting plasma glucose (mmol/L)/22.5. The citrate synthase activity was measured in the BAT using a colorimetric assay kit (BioVision, Milpitas, CA, USA). Hepatic glycogen was quantified according to the manufacturer’s instruction of a commercial kit (Sigma-Aldrich, USA). In addition, the lipid content was quantified in the liver after extraction with chloroform, as previously described [ 23 ]. Briefly, the tissues were homogenized in chloroform/methanol (2:1) solution. After 3 h of shaking, Milli-Q water was added, and the organic layer was separated by centrifugation (16,000 × g, 20 min) and dried overnight. Triglyceride concentration was measured as described above. 2.5. Immune Parameters One section of the colon and one section of the liver were homogenized in RIPA buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Sigma-Aldrich). The homogenate was centrifuged (12,000 × g, 10 min). In the supernatant from the colon and the liver, the levels IL-22 or IL-6 were, respectively, quantified with an ELISA kit (ELISA MAX TM ) Deluxe Set, Biolegend, San Diego, CA, USA). The values were normalized with the amount of protein quantified using the Bradford method. Plasma levels of IL-6 were measured using the Luminex ™ IL-6 Mouse Bead kit (Invitrogen, Waltham, MA, USA). In suspensions of the cecal content, secretory immunoglobulin A (sIgA) was measured using another commercial kit (Invitrogen, Waltham, MA, USA). Nutrients 2022,14, 440 4 of 15 2.6. Gene Expression Analyses Total RNA was isolated from different sections of the intestine (ileum and colon) using the commercial kit Nucleo Spin RNA (Macherey-Nagel, Nordrhein-Westfalen, Germany) and from the WAT and liver using the TRIsureTM reagent (Bioline, London, UK). Complementary DNA was prepared by the reverse transcription of 1 µ g of total RNA using the kit High-Capacity cDNA Reverse Transcription (Applied Biosystems, Foster City, CA, USA), following manufacturer ´ s instructions. The RT-qPCR was performed with the LightCycler ® 480 Instrument (Roche, Boulogne-Billancourt, France). The reaction consisted of LightCycler 480 SYBR Green I Master mix (Roche) and 300 nM of gene-specific primer pairs. Samples were run in duplicate, and the data were analyzed using the 2 −∆∆CT method. The qPCR program was previously described [ 23 ]. Targeted genes were normalized with the expression of ribosomal protein L19 (Rpl19), the housekeeping gene. Primer sequences are detailed in Table S2. 2.7. Histological Analysis The visceral WAT was stained with hematoxylin/eosin to quantify the size of the adipocytes. The bright-field digital images were taken using an E90I Nikon microscope (Nikon Corporation, Tokio, Japan) with an 8x objective, equipped with a digital camera (Nikon DS-5Mc). A combined analysis was carried using Fiji (ImageJ 1.49q Software, National Institutes of Health, Bethesda, MD, USA) and Nis Elements BR 3.2 software (Nikon Corporation, Japan). The cross-sectional area of each adipocyte was automatically recognized and calculated by the NIS-elements software. Artifacts were manually discarded. Three independent measurements from different sections were performed on each mouse. At least 350 adipocytes were quantified per measurement. 2.8. Gut Microbiota Composition Genomic DNA was extracted from the cecal content using a QIAamp PowerFecal DNA Kit (Qiagen, Hilden, Germany), and gut microbiota composition was analyzed by sequencing the V3-V4 hypervariable region of the 16S rRNA gene in an Illumina platform (MiSeq). The complete bioinformatics analysis is described in Supplementary Materials. 2.9. Statistical Analyses G*Power 3.1.9.2 was used to calculate sample size and GraphPad software (version 8, San Diego, CA, USA) for statistical analyses and plots. Shapiro–Wilk test was employed to assess data normality. For normally distributed data, differences were determined with oneway analysis of variance (ANOVA) or two-way ANOVA (as suitable) followed by post hoc Tukey’s multiple comparison tests. Welch’s correction was applied when variances were not equally distributed, followed by a Dunnett’s T3 multiple comparisons test. Non-normally distributed data were analyzed with the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. For gut microbiota analyses, Benjamini–Hochberg method was used to adjust p-values and Permutational Multivariate Analysis of Variance (adonis) followed by a pairwise post-hoc test (pairwiseAdonis). The results were considered statistically significant at p< 0.05. For all analyses, the Grubbs test was used for outlier detection. 3. Results 3.1. PTS Curbed Obesity by Influencing the White and Brown Adipose Tissue Metabolism in a Dose-Dependent Manner The HFHSD significantly increased body weight (Figure 1A) due to the expansion of the epididymal and inguinal WAT (Figure 1B). The administration of PTS curbed obesity in a dose-dependent manner; however, this effect only reached the statistical cut-off point at the higher dose. Accordingly, the histological analysis of the white fat pads showed that, at this dose, PTS prevented the increase in the adipocyte size induced by the HFHSD (Figure 1C,D). The morphological changes caused by the obesogenic diet were accompanied by clear increases in the expression of Itgax, coding for CD11c, a marker of M1 macrophages Nutrients 2022,14, 440 5 of 15 that are infiltrated in the adipose tissue in obesity [ 24 ] (Figure 1E). Only PTS at the higher dose reduced the expression of Itgax and Ccl2, coding for the chemokine MCP-1, which mediates the recruitment of macrophages. Nutrients 2022, 14, x FOR PEER REVIEW 5 of 15 3. Results 3.1. PTS Curbed Obesity by Influencing the White and Brown Adipose Tissue Metabolism in a Dose-Dependent Manner The HFHSD significantly increased body weight (Figure 1A) due to the expansion of the epididymal and inguinal WAT (Figure 1B). The administration of PTS curbed obesity in a dose-dependent manner; however, this effect only reached the statistical cut-off point at the higher dose. Accordingly, the histological analysis of the white fat pads showed that, at this dose, PTS prevented the increase in the adipocyte size induced by the HFHSD (Figure 1C,D). The morphological changes caused by the obesogenic diet were accompanied by clear increases in the expression of Itgax, coding for CD11c, a marker of M1 macrophages that are infiltrated in the adipose tissue in obesity [24] (Figure 1E). Only PTS at the higher dose reduced the expression of Itgax and Ccl2, coding for the chemokine MCP1, which mediates the recruitment of macrophages. Figure 1. (A) Body weight development (two-way ANOVA p < 0.001), (B) weight of the fat mass: eWAT (Kruskal–Wallis p < 0.01) and iWAT (one-way ANOVA p < 0.05), (C) histology of the visceral adipose tissue, (D) area of the adipocytes (one-way ANOVA p < 0.01), and (E) expression of markers of inflammation: Itgax (Kruskal–Wallis p < 0.01) and Ccl2 (Welch’s ANOVA p < 0.01). Mice were fed C A D Control HFHSD HFHSD + PTS low dose HFHSD + PTS high dose E Inflammatory markers B Body weight evolution 0 20406080 25 30 35 40 45 Days Body weight (g) a ac bc b Area of adipocytes 0 1000 2000 3000 4000 5000 Area (μm 2 ) bab a a BAT 0.0 0.2 0.4 0.6 0.8 Fat mass eWAT iWAT 0 2 4 6 8 %Body weight a bab a a bab ab Tnfa Ifng Ccl2 0 1 2 3 mRNA (relative expression) aa ab b Itgax 0 5 10 15 20 a b ab a Figure 1. ( A ) Body weight development (two-way ANOVA p< 0.001), ( B ) weight of the fat mass: eWAT (Kruskal–Wallis p< 0.01) and iWAT (one-way ANOVA p< 0.05), ( C ) histology of the visceral adipose tissue, ( D ) area of the adipocytes (one-way ANOVA p< 0.01), and ( E ) expression of markers of inflammation: Itgax (Kruskal–Wallis p< 0.01) and Ccl2 (Welch’s ANOVA p< 0.01). Mice were fed a control diet (n= 6–7); a high-fat, high-sucrose diet (HFHSD) (n= 6–8); and HFHSD with propyl propane thiosulfinate (PTS) at a low dose (0.1 mg/kg day) (n= 7–8) and a high dose (1 mg/kg day) (n= 7–8). Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple comparison tests for normally distributed data, except for body weight development, which was analyzed with a two-way ANOVA followed by Tukey’s post hoc test. Welch’s correction was applied when variances were not equally distributed. Non-normally distributed data were analyzed with the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Different superscript letters show statistical differences in the post hoc test when p< 0.05. Ccl2 gene codes for MCP-1; the Itgax gene codes for CD11c. We also analyzed the expression of molecular markers involved in fat mobilization (Figure 2A) and storage (Figure 2B). In the WAT, the HFHSD did not modify fat mobilization, Nutrients 2022,14, 440 6 of 15 but it significantly upregulated pathways related to fat uptake and storage, such as Lpl, Cd36, and Dgat2. In contrast, the effect of PTS on lipid metabolism was balanced, with reductions in either fat mobilization and storage (Figure 2A,B). Specifically, the higher dose of PTS reduced the expression of markers involved in catabolic processes such as lipolysis (Atgl,Hsl), and beta-oxidation (Cpt1a); and in anabolic processes such as fatty acid uptake (Lpl, Cd36), fatty acid synthesis (Acc1,Fas), and adipogenesis (Cebpb,Pparg). However, PTS did not counteract the decline in markers that were caused by the obesogenic diet (Supplementary Figure S1). All the changes attributed to PTS administration occurred without any differences in the water or food intake, or in the plasmatic levels of triglycerides, NEFA, or cholesterol (Table S3). Finally, we investigated whether the anti-obesogenic effect of PTS could be attributed to a higher thermogenic activity of the BAT. In agreement with our hypothesis, PTS increased the expression Cpt1a (the low and high dose) as well as Ppara and Prdm16 (at the higher dose) (Figure 2C). Hence, to confirm the capacity of PTS to increase the thermogenic activity of the BAT, we analyzed the mitochondrial activity by quantifying the citrate synthase activity. Accordingly, we found that PTS boosted this activity in a dose-dependent manner, which was only statistically different between the group treated with the highest dose of PTS versus the obese group (Figure 2D). 3.2. PTS Improved Systemic Glucose Homeostasis, Hepatic Metabolism and Inflammatory Response High-calorie diets are well-known to impair glucose metabolism. Here, we confirmed that mice fed an HFHSD had a worse response to the glucose tolerance test and exhibited uncontrolled insulinemia (Figure 3A,B). The administration of PTS tended to attenuate both effects in a dose-dependent manner. Moreover, even if fasting glycaemia and the hepatic glycogen levels remained unchanged between groups (Figures 3C and S2A), the metabolic disruption in response to the HFHSD led to a pre-diabetic state, as estimated by the HOMA-IR index, which was not observed in PTS-treated animals (Figure S2B). Moreover, the HFHSD triggered hepatic steatosis, as evidenced by an increase in the inflammatory tone (assessed by IL-6 quantification) (Figure 3D) and higher hepatic weight due to triglycerides accumulation (Figure 3E,F). No changes were detected in the plasmatic levels of IL-6 (Figure S2C). Meanwhile PTS at the highest dose significantly prevented both effects. Furthermore, the molecular analysis of the liver showed that the HFHSD upregulated lipid storage markers (Cd36,Dgat2, and Fas), which all remained at the same level as the control group in mice treated with the highest dose of PTS (Figure 3G). 3.3. PTS Had Minor Effects on the Intestinal Permeability and the Gut Barrier Function The in vivo assessment of intestinal permeability showed no differences caused either by diet or by PTS (Figure 4A). The HFHSD significantly altered the expression of genes involved in barrier function in the colon (Cldn3,Zo1, and Muc2) (Figure 4B), and ileum (Cldn3 and Ocln) (Figure S3A). In contrast, PTS at the highest dose restored the expression of Zo1 and Muc2 to the level of the control group (Figure 4B). Based on the role attributed to IL-22 in the gut barrier function [ 25 ], we measured this cytokine in the colon. The group fed an HFHSD presented a sharp drop in IL-22 levels, which the highest dose of PTS completely counteracted (Figure 4C). Additionally, in the colon, the HFHSD impaired the expression of the antimicrobial peptides (AMPs), known as Reg3g and DefA, both restored by the highest dose of PTS (Figure 4D). There were no changes in the expression of cell renewal markers in the colon (Figure 4D) or of AMPs in the ileum (Figure S3B). Lastly, in terms of the cecal content, we quantified the sIgA and observed an increase linked to the obesogenic diet, which was not prevented by PTS at either of the doses tested (Figure 4E). Nutrients 2022,14, 440 7 of 15 Nutrients 2022, 14, x FOR PEER REVIEW 7 of 15 Figure 2. In the white adipose tissue: Atgl (one-way ANOVA p < 0.01), Hsl (Kruskal–Wallis p < 0.001), Ppara (one-way ANOVA p < 0.01) and Cpt1a (Welch’s ANOVA p < 0.01); (A) expression of markers of lipid mobilization: Lpl (Welch’s ANOVA p < 0.0001), Cd36 (one-way ANOVA p < 0.001), Dgat2 (Welch’s ANOVA p < 0.0001), Acc1 (Kruskal–Wallis p < 0.001), Fas (Kruskal–Wallis p < 0.001), Cebpb (Kruskal–Wallis p < 0.01) and Pparg (Kruskal–Wallis p < 0.01); and (B) lipid storage. In the brown adipose tissue, (C) expression of fatty acid oxidation (FAO) and thermogenesis: Ppara (one-way ANOVA p < 0.05), Cpt1a (one-way ANOVA p < 0.01) and Prdm16 (Kruskal–Wallis p < 0.05); and (D) citrate synthase activity (one-way ANOVA p < 0.05). Mice were fed a control diet (n = 6–7); a highfat, high-sucrose diet (HFHSD) (n = 6–8); and HFHSD with propyl propane thiosulfinate (PTS) at a low dose (0.1 mg/kg day) (n = 6–8) and a high dose (1 mg/kg day) (n = 6–8). Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple comparison tests for normally distributed data, and Welch’s correction was applied when variances were not equally distributed. Non-normally distributed data were analyzed with the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Different superscript letters show statistical differences in the post hoc test when p < 0.05. C A B D Control HFHSD HFHSD + PTS low dose HFHSD + PTS high dose Lipid mobilization Lipid storage Atgl Hsl Ppara Cpt1a Ucp2 0.0 0.5 1.0 1.5 2.0 2.5 mRNA (relative expression) aa ab b ab a bc c a ab ab b ab a ab b FAO and Thermogenesis Ppara Cpt1a Ucp1 Prdm16 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 mRNA (relative expression) a b ab ab a a ab b ab b ab a Citrate synthase 0.0 0.5 1.0 1.5 2.0 mU/mg tissue ab a ab b Lpl Cd36 Dgat2 Acc1 Fas Cebpb Pparg 0.5 1.5 2.5 3.5 4.5 5.5 mRNA (relative expression) a b ac c a b a a a ab bc c a aab b ab b ab a a b ab a a b a a Figure 2. In the white adipose tissue: Atgl (one-way ANOVA p< 0.01), Hsl (Kruskal–Wallis p< 0.001), Ppara (one-way ANOVA p< 0.01) and Cpt1a (Welch’s ANOVA p< 0.01); ( A ) expression of markers of lipid mobilization: Lpl (Welch’s ANOVA p< 0.0001), Cd36 (one-way ANOVA p< 0.001), Dgat2 (Welch’s ANOVA p< 0.0001), Acc1 (Kruskal–Wallis p< 0.001), Fas (Kruskal–Wallis p< 0.001), Cebpb (Kruskal–Wallis p< 0.01) and Pparg (Kruskal–Wallis p< 0.01); and ( B ) lipid storage. In the brown adipose tissue, ( C ) expression of fatty acid oxidation (FAO) and thermogenesis: Ppara (one-way ANOVA p< 0.05), Cpt1a (one-way ANOVA p< 0.01) and Prdm16 (Kruskal–Wallis p< 0.05); and ( D ) citrate synthase activity (one-way ANOVA p< 0.05). Mice were fed a control diet (n= 6–7); a high-fat, high-sucrose diet (HFHSD) (n= 6–8); and HFHSD with propyl propane thiosulfinate (PTS) at a low dose (0.1 mg/kg day) (n= 6–8) and a high dose (1 mg/kg day) (n= 6–8). Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple comparison tests for normally distributed data, and Welch’s correction was applied when variances were not equally distributed. Non-normally distributed data were analyzed with the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Different superscript letters show statistical differences in the post hoc test when p< 0.05. Nutrients 2022,14, 440 8 of 15 Nutrients 2022, 14, x FOR PEER REVIEW 8 of 15 Figure 3. Systemic circulation: (A) Glucose tolerance test (two-way ANOVA p < 0.001); AUC (oneway ANOVA p < 0.05), (B) Insulinemia (one-way ANOVA p < 0.01), and (C) glycemia. In the liver: (D) hepatic IL6 levels (Welch’s ANOVA p < 0.001), (E) weight of the liver (one-way ANOVA p < 0.05), (F) triglycerides (Kruskal–Wallis p < 0.01), and (G) expression of markers of lipid metabolism: Cd36 (Welch’s ANOVA p < 0.01), Dgat2 (one-way ANOVA p < 0.05), Acc1 (one-way ANOVA p < 0.01), and Fas (one-way ANOVA p < 0.05). Mice were fed a control diet (n = 6–7); a high-fat, highsucrose diet (HFHSD) (n = 6–8); and HFHSD with propyl propane thiosulfinate (PTS) at a low dose (0.1 mg/kg day) (n = 7–8) and at a high dose (1 mg/kg day) (n = 7–8). Statistical analyses were performed by one-way analysis of variance (ANOVA), followed by post hoc Tukey’s multiple comparison tests for normally distributed data, and Welch’s correction was applied when variances were not equally distributed. Non-normally distributed data were analyzed with the Kruskal–Wallis test, followed by Dunn’s multiple comparisons test. Different superscript letters show statistical differences in the post hoc test when p < 0.05. “*” means significantly different within all groups, “#” means significantly different compared with the control group. ** p < 0.01; and # p < 0.05. 3.3. PTS Had Minor Effects on the Intestinal Permeability and the Gut Barrier Function The in vivo assessment of intestinal permeability showed no differences caused either by diet or by PTS (Figure 4A). The HFHSD significantly altered the expression of genes involved in barrier function in the colon (Cldn3, Zo1, and Muc2) (Figure 4B), and ileum (Cldn3 and Ocln) (Figure S3A). In contrast, PTS at the higher dose restored the expression of Zo1 and Muc2 to the level of the control group (Figure 4B). Based on the role attributed to IL-22 in the gut barrier function [25], we measured this cytokine in the colon. The group fed an HFHSD presented a sharp drop in IL-22 levels, which the highest dose of PTS completely counteracted (Figure 4C). Additionally, in the colon, the HFHSD impaired the expression of the antimicrobial peptides (AMPs), known as Reg3g and DefA, Figure 3. Systemic circulation: ( A ) Glucose tolerance test (two-way ANOVA p< 0.001); AUC (oneway ANOVA p< 0.05), ( B ) Insulinemia (one-way ANOVA p< 0.01), and ( C ) glycemia. In the liver: ( D ) hepatic IL6 levels (Welch’s ANOVA p< 0.001),( E ) weight of the liver (one-way ANOVA p< 0.05), ( F ) triglycerides (Kruskal–Wallis p< 0.01), and ( G ) expression of markers of lipid metabolism: Cd36 (Welch’s ANOVA p< 0.01), Dgat2 (one-way ANOVA p< 0.05), Acc1 (one-way ANOVA p< 0.01), and Fas (one-way ANOVA p< 0.05). Mice were fed a control diet (n= 6–7); a high-fat, high-sucrose diet (HFHSD) (n= 6–8); and HFHSD with propyl propane thiosulfinate (PTS) at a low dose (0.1 mg/kg day) (n= 7–8) and at a high dose (1 mg/kg day) (n= 7–8). Statistical analyses were performed by one-way analysis of variance (ANOVA), followed by post hoc Tukey’s multiple comparison tests for normally distributed data, and Welch’s correction was applied when variances were not equally distributed. Non-normally distributed data were analyzed with the Kruskal–Wallis test, followed by Dunn’s multiple comparisons test. Different superscript letters show statistical differences in the post hoc test when p< 0.05. “*” means significantly different within all groups, “#” means significantly different compared with the control group. ** p< 0.01; and #p< 0.05. Nutrients 2022,14, 440 9 of 15 Nutrients 2022, 14, x FOR PEER REVIEW 9 of 15 both restored by the highest dose of PTS (Figure 4D). There were no changes in the expression of cell renewal markers in the colon (Figure 4D) or of AMPs in the ileum (Figure S3B). Lastly, in terms of the cecal content, we quantified the sIgA and observed an increase linked to the obesogenic diet, which was not prevented by PTS at either of the doses tested (Figure 4E). Figure 4. Gut barrier function assessed (A) by FITC translocation in the systemic circulation, and (B) expression of tight junction markers and mucin in the colon: Cldn3 (one-way ANOVA p < 0.05), Zo1 (one-way ANOVA p < 0.05) and Muc2 (one-way ANOVA p < 0.0001), (C) levels of IL-22 in the colon (Kruskal–Wallis p < 0.01), and (D) expression of antimicrobial peptides and cell renewal markers: Reg3g (Kruskal–Wallis p < 0.01) and DefA (one-way ANOVA p < 0.05). In the cecal content, (E) levels of secretory immunoglobulin A (sIgA) (Kruskal–Wallis p < 0.05). Mice were fed a control diet (n = 6–7); a high-fat, high-sucrose diet (HFHSD) (n = 7–8); and HFHSD with propyl propane thiosulfinate (PTS) at a low dose (0.1 mg/kg day) (n = 7–8) and at a high dose (1 mg/kg day) (n = 7–8). Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple comparison tests. Non-normally distributed data were analyzed with the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Different superscript letters show statistical differences in the post hoc test when p < 0.05. 3.4. PTS Caused No Differences in Gut Microbiota Diversity and Only Minor Taxonomic Changes Non-metric multidimensional scaling (NMDS) showing the beta-diversity based on the Bray–Curtis dissimilarity indicated that the samples clustered based on the diet (control versus HFHSD) (Figure 5A). Accordingly, the three experimental groups that received the HFHSD presented a significant reduction in the Shannon index (Figure 5B) but no changes in the Chao1 diversity index (Figure 5C). When considering the changes in those OTUs identified at the genus level, the intake of HFHSD, independently of whether it was accompanied with the PTS treatment or not, caused decreases in Parabacteroides (Figure 5D) and increases in Bacteroides (Figure 5E) and Blautia (except at the high dose) (Figure 0 200 400 600 sIgA (ng/ml) a b ab ab DE CAB FITC-Dextran DefA Tcf4 Ki67 0 1 2 3 a b ab a Reg3g Antimicrobial peptides Cell renewal 0 500 1000 1500 FITC-Dextran (ug/ml) sIgA Gut barrier function markers Cldn3 Ocln Zo1 Muc2 0 1 2 3 4 5 mRNA (relative expression) a b ab a ab b ab ab a b aa 0 200 400 600 800 mRNA (relative expression) a b ab a IL-22 0 10 20 30 40 pg/g protein a b ab a Figure 4. Gut barrier function assessed ( A ) by FITC translocation in the systemic circulation, and ( B ) expression of tight junction markers and mucin in the colon: Cldn3 (one-way ANOVA p< 0.05), Zo1 (one-way ANOVA p< 0.05) and Muc2 (one-way ANOVA p< 0.0001), ( C ) levels of IL-22 in the colon (Kruskal–Wallis p< 0.01), and ( D ) expression of antimicrobial peptides and cell renewal markers: Reg3g (Kruskal–Wallis p< 0.01) and DefA (one-way ANOVA p< 0.05). In the cecal content, ( E ) levels of secretory immunoglobulin A (sIgA) (Kruskal–Wallis p< 0.05). Mice were fed a control diet (n= 6–7); a high-fat, high-sucrose diet (HFHSD) (n= 7–8); and HFHSD with propyl propane thiosulfinate (PTS) at a low dose (0.1 mg/kg day) (n= 7–8) and at a high dose (1 mg/kg day) (n= 7–8). Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple comparison tests. Non-normally distributed data were analyzed with the Kruskal– Wallis test followed by Dunn’s multiple comparisons test. Different superscript letters show statistical differences in the post hoc test when p< 0.05. 3.4. PTS Caused No Differences in Gut Microbiota Diversity and Only Minor Taxonomic Changes Non-metric multidimensional scaling (NMDS) showing the beta-diversity based on the Bray–Curtis dissimilarity indicated that the samples clustered based on the diet (control versus HFHSD) (Figure 5A). Accordingly, the three experimental groups that received the HFHSD presented a significant reduction in the Shannon index (Figure 5B) but no changes in the Chao1 diversity index (Figure 5C). When considering the changes in those OTUs identified at the genus level, the intake of HFHSD, independently of whether it was accompanied with the PTS treatment or not, caused decreases in Parabacteroides (Figure 5D) and increases in Bacteroides (Figure 5E) and Blautia (except at the high dose) (Figure 5F). Conversely, the administration of PTS at both doses was only associated with increases in the bacterial genus Intestimonas (Figure 5G). Additionally, the highest dose of PTS increased Alistipes (Figure 5H) and reduced the relative abundance of Bifidobacterium (Figure 5I).