nutrients Article Beneficial Effect of a Multistrain Synbiotic Prodefen® Plus on the Systemic and Vascular Alterations Associated with Metabolic Syndrome in Rats: The Role of the Neuronal Nitric Oxide Synthase and Protein Kinase A Pablo Llévenes 1 , Raquel Rodrigues-Díez 2,3,4 , Laia Cros-Brunsó 1 , Ma Isabel Prieto 4,5 , Laura Casaní 6 , Gloria Balfagón 1,3,4 and Javier Blanco-Rivero 1,3,4, * 1 Department of Physiology, School of Medicine, Universidad Autónoma de Madrid, Calle de Arzobispo Morcillo 4, 28029 Madrid, Spain; pablollevenes@gmail.com (P.L.); croslaia@gmail.com (L.C.-B.); gloria.balfagon@uam.es (G.B.) 2 Department of Pharmacology and Therapeutics, School of Medicine, Universidad Autónoma de Madrid, Calle de Arzobispo Morcillo 4, 28029 Madrid, Spain; raquel.rodrigues@uam.es 3 Center for Biomedical Research Network (CIBER) in Cardiovascular Diseases, Calle de Melchor Fernández Almagro 3, 28029 Madrid, Spain 4 Research Institute University Hospital la Paz (IdIPaz), Calle de Pedro Rico 6, 28029 Madrid, Spain; iprieto@intermic.com 5 Department of General and Digestive Surgery, Hospital Universitario la Paz, Paseo de la Castellana 261, 28046 Madrid, Spain 6 Research Institute of Santa Creu i Sant Pau Hospital, Carrer de Sant Quintí 77–79, 08041 Barcelona, Spain; lcasani@santpau.cat * Correspondence: javier.blanco@uam.es; Tel.: +34-91-497-5446  Received: 5 December 2019; Accepted: 18 December 2019; Published: 1 January 2020  Abstract: A high fat diet (HFD) intake is crucial for the development and progression of metabolic syndrome (MtS). Increasing evidence links gut dysbiosis with the metabolic and vascular alterations associated with MtS. Here we studied the use of a combination of various probiotic strains together with a prebiotic (synbiotic) in a commercially available Prodefen® Plus. MtS was induced by HFD (45%) in male Wistar rats. Half of the MtS animals received Prodefen® Plus for 4 weeks. At 12 weeks, we observed an increase in body weight, together with the presence of insulin resistance, liver steatosis, hypertriglyceridemia and hypertension in MtS rats. Prodefen® Plus supplementation did not affect the body weight gain but ameliorated all the MtS-related symptoms. Moreover, the hypertension induced by HFD is caused by a diminished both nitric oxide (NO) functional role and release probably due to a diminished neuronal nitric oxide synthase (nNOS) activation by protein kinase A (PKA) pathway. Prodefen® Plus supplementation for 4 weeks recovered the NO function and release and the systolic blood pressure was returned to normotensive values as a result. Overall, supplementation with Prodefen® Plus could be considered an interesting non-pharmacological approach in MtS. Keywords: metabolic syndrome; synbiotic; hypertension; superior mesenteric artery; perivascular nitrergic innervation; nitric oxide; neuronal nitric oxide synthase; protein kinase a 1. Introduction Obesity affected two billion people worldwide in 2015 with annual costs reaching two trillion USD [1]. The global prevalence has increased dramatically in the last four decades, reaching 10.8% Nutrients 2020, 12, 117; doi:10.3390/nu12010117 www.mdpi.com/journal/nutrients Nutrients 2020, 12, 117 2 of 18 in adult men and 14.9% in adult women [2]. Chronic consumption of a high-fat diet (HFD) induces overweight and is the main trigger for the development of metabolic syndrome (MtS) characterized by dyslipidemia, hypertension and impaired glucose homeostasis [3,4]. MtS is associated with high mortality and morbidity due to an increase in the prevalence of cardiovascular diseases, diabetes, chronic kidney diseases, cancer and musculoskeletal diseases [2]. The accumulation of adipose tissue promotes a pro-inflammatory and pro-oxidative microenvironment, leading to a chronic low-grade inflammation state [5]. Moreover, the secretion of adipokines from the adipose tissue alters the function of multiple vasoactive factors, increases peripheral vascular resistance and leads to hypertension. Evidence indicates that the blood flow and tissue perfusion required for cardiovascular system homeostasis is affected by nitric oxide (NO) synthesis [6,7]. Other factor implicated in the regulation of vascular resistance is perivascular innervation, specifically in certain vessels such as the mesenteric vascular bed, where blood flow is approximately 20–30% of the total cardiac output [8]. Previously we have reported that the neural control of mesenteric vasomotor tone is altered in rats fed on HFD for a short term, partly due to a decrease in neuronal NO release from perivascular nitrergic innervation [9,10]. Increasing evidence links the development of MtS and chronic low-grade inflammation to dysbiosis, an imbalance in the intestinal flora [11–13]. Some studies demonstrated that dysbiosis promotes the development of low-grade inflammation and consequently MtS, and others that dysbiosis is the result of low-grade inflammation during obesity and MtS. Either way, the modulation of dysbiosis by dietary strategies based on alterations of gut microbiome improves the health outcome of MtS [14]. The supplementation of probiotics, live bacterial strains, have been shown to modulate the alteration of gut microbiome [15] and to improve MtS symptoms, such as insulin resistance, lipid profile and high blood pressure in animal models [16,17]. It is considered that multistrain and/or multi-species probiotics have been shown in animal models to be more effective than monostrain probiotics [18]. Additionally, the use of prebiotics, non-digestible oligosaccharides that stimulates the growth of desired bacteria, together with a probiotic is more effective than probiotics alone by improving survival and implantation of live microbes in the gastrointestinal tract [19]. The commercial synbiotic formulation Prodefen® Plus combines various probiotic strains (Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium breve and Bifidobacterium infantis) together with a prebiotic, fructooligosaccharides. Both Prodefen® Plus, and a similar synbiotic commercial formula Prodefen® exert a beneficial effect in antibiotic-associated diarrhoea and acute gastroenteritis in children, respectively [20,21]. Regarding MtS, Prodefen® has shown an antihypertensive effect in spontaneously hypertensive rats [22]. Nevertheless, the possible beneficial effect of Prodefen® Plus in MtS remains unknown. Given the above, we hypothesized that a supplementation with the commercially available synbiotic formula Prodefen® Plus might improve the cardiometabolic alterations related to MtS, leading to a reduction in the administration of any pharmacological treatment. Hence, in the present study we aim to assess the effect of Prodefen® Plus in rats fed on HFD that develop MtS, with a special focus on the understanding of the vascular mechanisms implicated in the development of hypertension. 2. Materials and Methods 2.1. Animals and Diet All experimental procedures were approved by the Ethical Committee of the Universidad Autónoma de Madrid, and the Comunidad de Madrid (PROEX 322/16), are in compliance with NIH guidelines and follow the European Parliament Directive 2010/63/EU guidelines. Twenty-four male Wistar rats (initial weight 209.5 ± 3.1 g) were purchased from Harlan Ibérica SL, Barcelona, Spain and housed in the Animal Facility of the Universidad Autónoma de Madrid (Registration number EX-021U). Rats were held in groups of 2 in appropriate cages in controlled environmental conditions Nutrients 2020, 12, 117 3 of 18 Nutrients 2020, 12, x FOR PEER REVIEW 3 of 19 24 °C, 55% relative humidity and 12 h light-dark cycle). All rats had access to drinking water and (20–24 ◦ C, 55% relative humidity and 12 h light-dark cycle). All rats had access to drinking water and specific rat chow ad libitum. specific rat chow ad libitum. Animals were randomly divided into three groups: (I) Rats fed a commercial standard diet (4.0% Animals were randomly divided into three groups: (I) Rats fed a commercial standard diet (4.0% fat; 2.9 Kcal/g; Tekland global rodent diet 2014, ENVIGO, Huntingdon, UK) for 12 weeks as a control fat; 2.9 Kcal/g; Tekland global rodent diet 2014, ENVIGO, Huntingdon, UK) for 12 weeks as a control group (CT; n = 8); (II) rats with metabolic syndrome (MtS, n = 8), fed a HFD (45% fat; 4.6 Kcal/g; 58V8, group (CT; n = 8); (II) rats with metabolic syndrome (MtS, n = 8), fed a HFD (45% fat; 4.6 Kcal/g; 58V8, TestDiet, Columbia, USA) for 12 weeks; and (III) rats with MtS, fed a HFD (45% fat, 4.6 Kcal/g) for 12 TestDiet, Columbia, USA) for 12 weeks; and (III) rats with MtS, fed a HFD (45% fat, 4.6 Kcal/g) for weeks, supplemented with the multistrain synbiotic Prodefen® ® Plus (adjusted doses to 107 colony 12 weeks, supplemented with the multistrain synbiotic Prodefen Plus (adjusted doses to 107 colony forming units (c.f.u.)/day) for the last 4 weeks (MtS-SYNB, n = 8). forming units (c.f.u.)/day) for the last 4 weeks (MtS-SYNB, n = 8). Prodefen®®Plus (formulation which combines 990 mg of fructooligosaccharides with various Prodefen Plus (formulation which combines 990 mg of fructooligosaccharides with various probiotic strains −101010c.f.u. Lactobacillus rhamnosus LGG and 1099c.f.u. of a mixture of: Lactobacillus probiotic strains −10 c.f.u. Lactobacillus rhamnosus LGG and 10 c.f.u. of a mixture of: Lactobacillus casei, Streptococcus thermophilus, Bifidobacterium brevis, Lactobacillus acidophilus, Bifidobacterium infantis casei, Streptococcus thermophilus, Bifidobacterium breve, Lactobacillus acidophilus, Bifidobacterium infantis and Lactobacillus bulgaricus), was generously provided by Italfarmaco S.A. This synbiotic formula was and Lactobacillus bulgaricus), was generously provided by Italfarmaco S.A. This synbiotic formula was administered to the rats dissolved in drinking water, once the obesity had been stablished [9,10]. The administered to the rats dissolved in drinking water, once the obesity had been stablished [9,10]. dose (107 c.f.u./day) and administration time (4 weeks) of Prodefen® Plus was chosen based on The dose (107 c.f.u./day) and administration time (4 weeks) of Prodefen® Plus was chosen based on preliminary pilot studies, choosing the lowest dose/time in which we found a systemic effect. The preliminary pilot studies, choosing the lowest dose/time in which we found a systemic effect. The body body weight was measured every two weeks. During the last 4 weeks, the water intake was measured weight was measured every two weeks. During the last 4 weeks, the water intake was measured every every two days to assure the intake of appropriate dose of the synbiotic. The summary of the two days to assure the intake of appropriate dose of the synbiotic. The summary of the experimental experimental procedure is represented in Figure 1. procedure is represented in Figure 1. Dietary Synbiotic End of intervention supplementation procedure BW BW BW GTT GTT GTT SBP SBP SBP BW BW BW BW Weeks 0 2 4 6 8 10 12 Figure 1. Diagrammatic representation of the 12-weeks experimental procedure. Abbreviations: body weight1.(BW), Figure glucose tolerance Diagrammatic test (GTT), representation systolic of the blood 12-weeks pressure (SBP). experimental procedure. Abbreviations: body weight (BW), glucose tolerance test (GTT), systolic blood pressure (SBP). 2.1.1. Glucose Tolerance Test 2.1.1. The Glucose Tolerance oral glucose Test test (GTT) was performed at 0, 8 and 12 weeks according to a standard tolerance protocol [23,24]. The oral After glucose an overnight tolerance fasting, test (GTT) wasa performed single oral at dose0, 8(2and g/kg12of bodyaccording weeks weight) oftoglucose was a standard delivered. Blood glucose was then measured from the tail vein just before, and at 15, 30, protocol [23,24]. After an overnight fasting, a single oral dose (2 g/kg of body weight) of glucose was 60, 90 and 120 min after glucose intake, ® delivered. Blood glucose wasusing then test strips and measured fromreader (FreeStyle the tail vein just Optium before, ,and Abbot Laboratories at 15, S.A., 30, 60, 90 and Madrid, Spain) (Figure 1). 120 min after glucose intake, using test strips and reader (FreeStyle Optium , Abbot Laboratories ® S.A., Madrid, Spain) (Figure 1). 2.1.2. Systolic Blood Pressure 2.1.2. The systolic Systolic blood Blood pressure was measured at 0, 8 and 12 weeks in awake rats by a tail-cuff method, Pressure using a caudal artery plethysmograph (NIPREM 645, Cibertec S.A., Madrid, Spain) as published The systolic blood pressure was measured at 0, 8 and 12 weeks in awake rats by a tail-cuff previously [9]. (Figure 1) method, using a caudal artery plethysmograph (NIPREM 645, Cibertec S.A., Madrid, Spain) as published 2.2. Animalpreviously [9]. Euthanasia, (Figure Sample 1) Collection and Sample Analysis After Euthanasia, 2.2. Animal an overnight fasting, Sample rats were Collection anesthetized and Sample Analysis(100 mg/kg ketamine hydrochloride and 12 mg/kg Xylazine; i.m.) and euthanized by exsanguination by the infrahepatic inferior cava vein After an puncture. overnight Visceral fasting, rats white and epididymal were anesthetized adipose pads(100 weremg/kg ketamine collected hydrochloride for posterior andLeft dissection. 12 mg/kg Xylazine; i.m.) and euthanized by exsanguination tibia length was measured as a parameter to evaluate body weight.by the infrahepatic inferior cava vein puncture. Visceral and epididymal white adipose pads were collected for posterior dissection. Left tibia length was measured as a parameter to evaluate body weight. Nutrients 2020, 12, 117 4 of 18 2.2.1. Liver Histology Liver was extracted, rinsed in a saline solution and weighed. The left lateral liver lobe was cryoprotected (30% w/v sucrose in phosphate saline buffer) and frozen at −70 ◦ C until use. Liver samples were embedded in optimum cutting temperature compound (OCT Tissue Tek, Sigma-Aldrich, Madrid, Spain), 5 µm cryostat sections were stained with lipid dye Sudan III and random images were taken at 20× magnification. Quantification was performed using Image-Pro Plus 7 software (Media Cybernetics, MD, USA). 2.2.2. Serum Biochemical Parameters Blood samples were kept at room temperature for 2 h and were centrifuged afterwards (2000× g 15 min, 4 ◦ C). The serum was then collected and kept at −70 ◦ C until use. Serum concentration of total cholesterol (TC), triglycerides (TG) and high density lipoproteins (HDL) were measured by biochemical analyser RAL LC5 (Barcelona, Spain). Low density lipoprotein (LDL) levels were indirectly calculated using the Friedewald formula: LDL = TC-((TG/5) + HDL) as published previously [25]. 2.2.3. Vascular Reactivity The superior mesenteric artery was carefully dissected, cleaned of connective tissue and maintained at 4 ◦ C in Krebs-Henseleit solution (in mmol/L: 115 NaCl, 25 NaHCO3 , 4.7 KCl, 1.2 MgSO4 .7H2 O, 2.5 CaCl2 , 1.2 KH2 PO4 , 11.1 glucose, and 0.01 Na2 EDTA) bubbled with a 95% O2 5% CO2 mixture until use. The segments were endothelium-denuded by gently rubbing the luminal surface of the segments with a thin wooden stick to eliminate the main source of vasoactive substances. Some segments were quickly frozen in liquid nitrogen and maintained at −70 ◦ C. Vascular reactivity experiments were performed in endothelium denuded segments of 2 mm length according to previously published method [26]. Briefly, two parallel stainless-steel pins were introduced through the lumen of the vascular segment: one was fixed to the bath wall and the other connected to a force transducer (Grass FTO3C; Quincy, MA, USA) that was connected to a model 7D Grass polygraph. For electric field stimulation (EFS) experiments, segments were mounted between two platinum electrodes 0.5 cm apart and connected to a stimulator (Grass, model S44) modified to supply the appropriate current strength. Segments were suspended in an organ bath containing 5 mL of KHS at 37 ◦ C continuously bubbled with a 95% O2 5% CO2 mixture (pH 7.4). The segments were subjected to a tension of 4.9 mN, which was readjusted every 15 min during a 90 min equilibration period before drug administration. After this, the vessels were exposed to 75 mmol/L KCl to confirm their functional integrity. The absence of vascular endothelium after the washout period was tested by the inability of 10 µmol/L acetylcholine (ACh) to relax segments precontracted with noradrenaline. Endothelium removal did not alter the contractions elicited by KCl [10,23]. Frequency-response curves to EFS (1, 2, 4, 8 and 16 Hz) were performed. The parameters used for EFS were 200 mA, 0.3 ms, 1–16 Hz, for 30 s with an interval of 1 min between each stimulus, the time required to recover the basal tone. A washout period of at least 1 h was necessary to avoid desensitisation between consecutive curves. Two successive frequency-response curves separated by 1 h intervals produced similar contractile responses [26]. To analyse the participation of NO in the EFS-induced response in our experimental procedure, 0.1 mmol/L Nω-nitro-L-arginine methyl ester (L-NAME), a non-specific inhibitor of nitric oxide synthase, was added to the bath 30 min before performing the second frequency–response curve. The vasodilator response to the NO donor, diethylamine NONOate, (DEA-NO, 0.1 nmol/L–0.1 mmol/L was determined in noradrenaline-precontracted segments from all experimental groups. 2.2.4. Nitric Oxide Release Nitric oxide (NO) release experiments were performed in endothelium-denuded mesenteric segments using the fluorescence probe 4,5-diaminofluorescein (DAF-2) as previously described [9,10,27]. Nutrients 2020, 12, 117 5 of 18 Briefly, segments from all the experimental groups were subjected to an equilibration period of 60 min in HEPES buffer (in mmol/L: NaCl 119; HEPES 20; CaCl2 1.2; KCl 4.6; MgSO4 1; KH2 PO4 0.4; NaHCO3 5; glucose 5.5; Na2 HPO4 0.15; pH 7.4) at 37 ◦ C. Afterwards, arteries were transferred to a HEPES-filled 400 µL chamber and incubated with 2 µmol/L DAF-2 for 30 min. Then the medium was collected to measure basal NO release. Once the organ bath was refilled, cumulative EFS pulses of 30 s (1, 2, 4, 8 and 16 Hz) were applied at 1 min intervals. The fluorescence of the medium was measured at room temperature using a spectrofluorometer (Jenway 6280 Fluorimeter) with excitation wavelength set at 492 nm and emission wavelength at 515 nm. Some segments were incubated with 1 µmol/L H89 (a PKA (protein kinase A) inhibitor), 0.1 µmol/L calfostin C (a PKC (protein kinase C) inhibitor), or 10 µmol/L LY 294002 (a phosphatidylinositol 3-kinase -PI3K- inhibitor). The EFS-induced NO release was calculated by subtracting basal NO release from that evoked by EFS. Furthermore, blank samples were collected in the same way from segment-free medium in order to subtract background emission. The amount of NO released was expressed as arbitrary fluorescence units (A.F.U.)/mg tissue. 2.2.5. PKA and PKC Activity Assays PKA and PKC activities were determined using a PKA and PKC kinase activity assay kit (Abcam, Cambridge, UK), respectively following the manufacturers’ protocols. Briefly, frozen arteries from all experimental groups were homogenised in a lysis buffer (0.01 mmol/L Tris-HCl, 1 mmol/L sodium vanadate, 1% SDS, pH 7.4), and centrifuged at 12,000× g for 10 min at 4 ◦ C. The supernatant was then collected and used for the assay. Protein content was measured using a DC protein assay kit (BioRad, Madrid, Spain). Results were expressed as optical density (O.D.) units/µg protein. 2.2.6. Western Blot Western blot analysis was performed as previously described [8,9]. Briefly, arteries were homogenized, and 30 µg protein was loaded in each lane. A mouse monoclonal antibody against neuronal nitric oxide synthase (nNOS, 1:2000, BD Biosciences), a rabbit polyclonal anti-pan-AKT antibody (1:1000, Abcam), or a rabbit polyclonal anti-pan-AKT (phospho T308, 1:500, Abcam) were used. The development and quantification of the images were performed using Quantity One software (Windows v4.6.6, Bio-Rad, Madrid, Spain). The same membrane was used to correct protein expression in each sample, by means of a monoclonal anti-β-actin−peroxidase antibody (1:50,000; Sigma-Aldrich, Madrid, Spain). 2.2.7. Detection of Superoxide Anions Superoxide anion levels were measured using lucigenin chemiluminescence, as previously described [10,27]. Briefly, endothelium-denuded segments from all experimental groups were equilibrated for 30 min in HEPES buffer at 37 ◦ C, transferred to test tubes that contained 1 mL HEPES buffer (pH 7.4) with lucigenin (5 µmol/L) and then kept at 37 ◦ C. The luminometer (Optocom I, GEM Biomedical Inc., Hamden, CT, USA) was set to report arbitrary units of emitted light; repeated measurements were collected (10 s intervals for 5 min) and averaged. 4,5-Dihydroxy-1,3-benzene-disulphonic acid “Tiron” (10 mmol/L), a cell-permeant, non-enzymatic superoxide anion scavenger, was added to quench the superoxide anion-dependent chemiluminescence. Blank samples (HEPES without arterial segment) were collected in the same way from culture medium without mesenteric segments to subtract background emission. 2.3. Drugs Used L-Noradrenaline hydrochloride, ACh chloride, diethylamine NONOate diethylammonium salt, Nω-nitro-L-arginine methyl ester (L-NAME) hydrochloride, lucigenin, tiron, DAF-2, calfostin C, H89, and LY294002 were used. All drugs were purchased from Sigma-Aldrich (Spain) except for LY294002 and H89, which were obtained from Tocris (Spain). Stock solutions (10 mmol/L) of drugs were made Nutrients 2020, 12, x FOR PEER REVIEW 6 of 19 Nutrients 2020, 12, 117 6 of 18 in distilled water, except for NA, which was dissolved in a NaCl (0.9%)–ascorbic acid (0.01% w/v) solution, or DAF, H89 and calfostin C and LY294002, which were dissolved in dimethyl sulfoxide. in distilled water, except for NA, which was dissolved in a NaCl (0.9%)–ascorbic acid (0.01% w/v) solution, 2.4. Data or DAF, H89 and calfostin C and LY294002, which were dissolved in dimethyl sulfoxide. Analysis Graph 2.4. Data representation and statistical analysis were performed using GraphPad Prism 6.0 Analysis software (California, USA). The adipose tissue pads and liver weight were normalised using tibia Graph representation and statistical analysis were performed using GraphPad Prism 6.0 software length. The responses induced by EFS were expressed as a % of the initial contraction elicited by (CA, USA). The adipose tissue pads and liver weight were normalised using tibia length. The responses 75 mmol/L KCl. To compare the effect of preincubation with L-NAME in EFS-induced contraction induced by EFS were expressed as a % of the initial contraction elicited by 75 mmol/L KCl. To compare experiments, differences between areas under the curve (dAUC) were analysed. The relaxation the effect of preincubation with L-NAME in EFS-induced contraction experiments, differences between induced by DEA-NO was expressed as a % of the initial contraction elicited by NA. Results were areas under the curve (dAUC) were analysed. The relaxation induced by DEA-NO was expressed expressed as mean ± S.E.M. The body weight evolution, the glucose tolerance test and the vasomotor as a % of the initial contraction elicited by NA. Results were expressed as mean ± S.E.M. The body responses to DEA-NO or EFS were compared by means of an unpaired two-way analysis of variance weight evolution, the glucose tolerance test and the vasomotor responses to DEA-NO or EFS were (ANOVA). When comparing the effect of L-NAME on the EFS-induced contraction, a paired two- compared by means of an unpaired two-way analysis of variance (ANOVA). When comparing the way ANOVA was used. For the body weight gain, food and water intake, lipid profile, blood effect of L-NAME on the EFS-induced contraction, a paired two-way ANOVA was used. For the body pressure, KCl, dAUC, NO, superoxide anion, PKA activity, PKC activity and Western blot weight gain, food and water intake, lipid profile, blood pressure, KCl, dAUC, NO, superoxide anion, densitometry analyses, a Shapiro-Wilk test was applied to confirm the normality of the population PKA activity, PKC activity and Western blot densitometry analyses, a Shapiro-Wilk test was applied to data, followed by a one-way ANOVA with a Newman–Keuls post-hoc test. p < 0.05 was considered confirm the normality of the population data, followed by a one-way ANOVA with a Newman–Keuls significant. post-hoc test. p < 0.05 was considered significant. 3. Results 3. Results 3.1. The 3.1. The Effect EffectofofHFD HFDand andSynbiotic SynbioticSupplementation Supplementationon onthe theBody BodyWeight WeightGain Gainand andLipid LipidProfile Profile Inthe In thepresent presentstudy studyweweinduced induced obesity obesity in in Wistar Wistarrats rats by by the the administration administration ofof aa HFD HFD (45% (45% fat). fat). The obesity The obesity was wasestablished established after after 88weeks. weeks. At At week week eight, eight, eight eight rats rats out out of of 16 16 fed fed onon aa HFD HFD were were administered a commercial synbiotic supplement Prodefen®® Plus. After 12 weeks, the body weight administered a commercial synbiotic supplement Prodefen Plus. After 12 weeks, the body weight increasewas increase wassignificantly significantlyhigher higherininMtS MtSrats ratscompared comparedtotoCT.CT.The Thesupplementation supplementationofofProdefen ®®Plus Prodefen Plus didnot did notaffect affectthe thebody bodyweight weightininMtS MtSrats rats(MtS-SYNB) (MtS-SYNB)(Figure (Figure2). 2). Figure 2. Body weight evolution in CT (control group), MtS (metabolic syndrome) and MtS-SYNB Figure 2. Body weight evolution in CT (control group), MtS (metabolic syndrome) and MtS-SYNB (MtS rats supplemented with Prodefen®Plus) groups over 12 weeks. Body weight is expressed as (MtS rats supplemented with Prodefen® Plus) groups over 12 weeks. Body weight is expressed as mean ± S.E.M. Statistical analysis: Unpaired two-way ANOVA. * p < 0.05 CT vs. MtS; + p < 0.05 CT vs. mean ± S.E.M. Statistical analysis: Unpaired two-way ANOVA. * p < 0.05 CT vs. MtS; + p < 0.05 CT vs. MtS-SYNB. n = eight animals per group. MtS-SYNB. n = eight animals per group. The body weight increase in MtS and MtS-SYNB group was accompanied by an increase in The body weight increase in MtS and MtS-SYNB group was accompanied by an increase in epididymal and visceral adipose pads. The food intake in MtS and MtS-SYNB group was lower and epididymal and visceral adipose pads. The food intake in MtS and MtS-SYNB group was lower and conversely the calories intake was higher. Water intake was comparable among groups (Table 1). conversely the calories intake was higher. Water intake was comparable among groups (Table 1). Nutrients 2020, 12, 117 7 of 18 Table 1. Body weight gain, food and water intake and lipid profile. Parameters CT MtS MtS-SYNB Body weight increase 1 (%) 87.8 ± 2.1 111.3 ± 7.1 * 106.1 ± 9.1 + Epididymal adipose pad 2 (g/cm) 1.66 ± 0.08 3.30 ± 0.34 * 3.44 ± 0.33 + Visceral adipose pad 2 (g/cm) 1.39 ± 0.06 3.05 ± 0.35 * 3.54 ± 0.30 + Liver weight 2 (g/cm) 1.91 ± 0.07 2.01 ± 0.09 1.86 ± 0.09 Food intake 3 (g/day) 20.96 ± 0.47 16.48 ± 0.47 * 16.67 ± 0.86 + Calorie intake 3 (Kcal/day) 60.75 ± 1.34 75.80 ± 2.14 * 76.69 ± 3.93 + Water intake 3 (mL/day) 36.59 ± 2.08 35.91 ± 1.02 38.16 ± 0.76 TC 4 (mg/dL) 88.88 ± 5.07 81.8 ± 5.37 90.38 ± 5.93 TG 4 (mg/dL) 112.20 ± 55.80 156.00 ± 16.37 * 115.40 ± 6.89 # HDL 4 (mg/dL) 37.29 ± 2.32 30.37 ± 2.19 35.41 ± 2.57 LDL 4 (mg/dL) 20.61 ± 2.47 25.31 ± 3.53 31.89 ± 3.08 Notes: Data are expressed as mean ± S.E.M. Statistical analysis: One-way ANOVA followed by a Newman–Keuls post-hoc test. * p < 0.05 CT vs. MtS; # p < 0.05 MtS vs. MtS-SYNB; + p < 0.05 CT vs. MtS-SYNB. n = 8 animals per group. 1 Body weight increase is expressed as % of the initial body weight. 2 Respective weights were normalized to tibia length (in cm; CT: 5.25 ± 0.03; MtS: 5.26 ± 0.09; MtS-SYNB: 5.19 ± 0.09). 3 Food and water intake were measured every two days in the last 4 weeks of the experimental procedure coinciding with the administration of the synbiotic. 4 Serum total cholesterol (TC), triglycerides /TG), high density lipoproteins (HDL) and low density lipoproteins (LDL) were analysed at the end of the experimental period of 12 weeks. CT (control group), MtS (metabolic syndrome), MtS rats supplemented with Prodefen®Plus (MtS-SYNB). Total cholesterol, LDL and HLD levels did not differ between groups, while the levels of plasma TG were significantly increased in the MtS group as compared to CT group and were returned to control levels in MtS-SYNB group (Table 1). Given the fact that increased TG levels leads to a development of liver steatosis, we next analysed the presence of lipids in liver sections by Sudan III lipid dye. Even though liver weight was comparable among groups, the HFD led to a development of steatosis as demonstrated by the presence of lipid vacuoles throughout the hepatic left lateral lobe. Interestingly, supplementation of Prodefen® Plus decreased the level of steatosis, even though not to the control state (Figure Nutrients 3). 2020, 12, x FOR PEER REVIEW 8 of 19 3. Liver Figure Figure sections 3. Liver were sections stained were stainedwith with Sudan III lipid Sudan III lipiddye. dye.Representative Representative (20 × (20 × imagesimages magnification) fromfrom magnification) CT (a), MtS CT (a), MtS(b) (b)and MtS-SYNB and MtS-SYNB (c)(c) ratsrats are shown. are shown. The The scale barscale bar represents represents 100 100 µm.μm.(d)(d)Quantitative Quantitative analysis analysisof Sudan of SudanIII lipid III dye staining. lipid Results areResults dye staining. represented are as % of stainedas % of represented stained area area(mean (mean ± S.E.M). Statistical analysis: One-way ANOVA followed by a Newman–Keuls post-hoc ± S.E.M). Statistical analysis: One-way ANOVA followed by a Newman–Keuls test. * p < 0.05 CT vs. MtS; # p < 0.05 MtS vs. MtS-SYNB. post-hoc test. * p < 0.05 CT vs. MtS; # p < 0.05 MtS vs. MtS-SYNB. 3.2. The Effect of Synbiotic Supplementation on MtS Induced Alterations MtS is related to several metabolic alterations, therefore next we analysed the effect of HFD and synbiotic supplementation on glucose homeostasis. Although basal glycemia was comparable among groups, insulin resistance was developed already at week eight of HFD and was maintained until week 12. The insulin resistance was ameliorated by Prodefen® Plus supplementation, even though not Figure 3. Liver sections were stained with Sudan III lipid dye. Representative images (20 × magnification) from CT (a), MtS (b) and MtS-SYNB (c) rats are shown. The scale bar represents 100 μm. (d) Quantitative analysis of Sudan III lipid dye staining. Results are represented as % of stained Nutrients 2020, area 12, 117 (mean ± S.E.M). Statistical analysis: One-way ANOVA followed by a Newman–Keuls post-hoc 8 of 18 test. * p < 0.05 CT vs. MtS; # p < 0.05 MtS vs. MtS-SYNB. 3.2. The Effect of Synbiotic Supplementation on MtS Induced Alterations 3.2. The Effect of Synbiotic Supplementation on MtS Induced Alterations MtS is related to several metabolic alterations, therefore next we analysed the effect of HFD and MtS is related to several metabolic alterations, therefore next we analysed the effect of HFD and synbiotic supplementation on glucose homeostasis. Although basal glycemia was comparable among synbiotic supplementation on glucose homeostasis. Although basal glycemia was comparable among groups, insulin resistance was developed already at week eight of HFD and was maintained until week groups, insulin resistance was developed already at week eight of HFD and was maintained until 12. The insulin resistance was ameliorated by Prodefen® Plus supplementation, even though not to the week 12. The insulin resistance was ameliorated by Prodefen® Plus supplementation, even though not control state (Figure 4). to the control state (Figure 4). Figure 4. Evolution of insulin resistance measured by glucose tolerance test in CT, MtS and MtS-SYNB Figure rats 4. Evolution at the beginning of of insulin resistance procedure measured (a), after 8 weeks ofbydiet glucose tolerance(b) intervention test in CT, and afterMtS and MtS-SYNB 12 weeks once the rats at thesupplementation synbiotic beginning of procedure (a), (c). terminated afterResults 8 weeks ofexpressed are diet intervention (b) and after as mg glucose/dL 12 weeks (mean once ± S.E.M). the synbiotic Statistical supplementation analysis: terminated Unpaired two-way ANOVA. * p < 0.05 (c). Results areCT expressed vs. MtS;as < 0.05 # pmg glucose/dL MtS vs. (mean ± S.E. MtS-SYNB; +M). p < Statistical 0.05 CT vs.analysis: MtS-SYNB. Unpaired two-way ANOVA. * p < 0.05 CT vs. MtS; # p < 0.05 MtS vs. MtS- SYNB; + p < 0.05 CT vs. MtS-SYNB. Hypertension is also related to MtS, therefore next we measured the systolic blood pressure using tail cuff method. At week 0 all the animals showed a normotensive phenotype. At week eight, animals fed on a HFD showed an increased systolic blood pressure reaching levels considered as hypertension. The supplementation of Prodefen® Plus for 4 weeks normalized the systolic blood pressure reaching levels comparable to the control group (Table 2). Table 2. Systolic blood pressure. CT MtS MtS-SYNB Week 0 1 118.5 ± 1.8 110.9 ± 3.0 * 110.1 ± 2.7 + Week 8 2 119.5 ± 2.4 141.1 ± 1.6 * 137.4 ± 1.0 + Week 12 3 120.3 ± 3.7 145.3 ± 2.1 * 120.6 ± 1.6 # Notes: Results are expressed as mmHg (mean ± S.E.M). Statistical analysis: One-way ANOVA followed by a Newman–Keuls post-hoc test. * p < 0.05 CT vs. MtS; # p < 0.05 MtS vs. MtS-SYNB; + p < 0.05 CT vs. MtS-SYNB. n = eight animals per group.1 Beginning of procedure. 2 Eight weeks of dietary intervention. 3 Twelve weeks of dietary intervention (4 weeks of synbiotic supplementation in MtS + SYNB group). 3.3. Mechanism of HFD-Induced Hypertension 3.3.1. Nitric Oxide Release The alterations in blood pressure are partially linked to modifications in peripheral vascular resistance. Among the multiple vasoactive factors which regulate vascular tone in superior mesenteric artery, NO has a relevant role, acting as a potent vasodilator. Given the great relevance of nitrergic innervation in the regulation of mesenteric resistance, we analysed the release of neuronal NO release in segments from all experimental groups. The application of an EFS pattern induced NO release in mesenteric segments without endothelium from all groups. As expected, NO release was significantly Nutrients 2020, 12, 117 9 of 18 decreased in MtS group as compared to CT group. Interestingly, the supplementation with Prodefen® Plus for 4 weeks recovered the NO release levels comparable to the control group (Table 3). Table 3. Nitric oxide and superoxide anion release in endothelium-denuded mesenteric rings. CT MtS MtS SYNB NO release (A.F.U./mg tissue) 1 2518 ± 147 1065 ± 10 * 2498 ± 279 # Superoxide anion 55.26 ± 3.01 55.78 ± 8.13 55.74 ± 6.54 release(C.U./min/mg tissue) 2 Notes: 1 Results are expressed as arbitrary fluorescence units (A.F.U.)/mg tissue. Mean ± S.E.M. Statistical analysis: One-way ANOVA followed by a Newman–Keuls post-hoc test. * p < 0.05 CT vs. MtS; # p < 0.05 MtS vs. MtS-SYNB. n = five to eight animals per group.2 Results are expressed as chemiluminescence units (C.U.)/minute/mg tissue. Mean ± S.E.M. n = five to eight animals per group. 3.3.2. Functional Role for Neuronal Nitric Oxide: Vascular Function Alteration in blood pressure is linked to modifications in smooth muscle sensitivity to nitric oxide (NO). However, in our model we did not find any differences in the vasodilator response to NO donor DEA-NO among Nutrients 2020, 12, x groups (Figure FOR PEER REVIEW5a). 10 of 19 Figure 5. (a) Vasodilator response to DEA-NO in mesenteric segments from CT, MtS and MtS-SYNB rats. Results Figure 5. (a)are expressed response Vasodilator as % of relaxation to DEA-NO elicited by NA (insegments in mesenteric from ± mN: CT: 11.81 CT,1.1;MtS MtS: 12.36 and ± 1.03; MtS-SYNB MtS-SYNB: 14.41 rats. Results ± 1.28). Mean are expressed as %±ofS.E.M. (b) Vasoconstrictor relaxation elicited by NAresponse to EFS (in mN: CT: in ±mesenteric 11.81 segments 1.1; MtS: 12.36 ± 1.03; from CT, MtS and MtS-SYNB rats. Results are expressed as a % of previous contraction MtS-SYNB: 14.41 ± 1.28). Mean ± S.E.M. (b) Vasoconstrictor response to EFS in mesenteric segments elicited by KCI (in mN: CT: 13.03 ± 0.74; MtS: 11.55 ± 0.51; MtS-SYNB: 12.45 ± 0.72). Mean ± S.E.M. Statistical from CT, MtS and MtS-SYNB rats. Results are expressed as a % of previous contraction elicited by analysis: Unpaired two-way KCI (in mN: ANOVA. CT: 13.03 ± 0.74; < 0.0511.55 * p MtS: CT vs. # p < 0.05 MtS vs. MtS-SYNB. MtS;MtS-SYNB: ± 0.51; 12.45 ± 0.72). Mean ± S.E.M. Statistical analysis: Unpaired two-way ANOVA. * p < 0.05 CT vs. MtS; # p < 0.05 MtS vs. MtS-SYNB. Next, we aimed to determine whether the observed alteration in NO release have a relevant functional Next, role. we aimedWe toapplied EFSwhether determine to inducethe aobserved frequency-dependent alteration in NO contractile response release have in a relevant endothelium-denuded functional role. We applied EFS to induce a frequency-dependent contractile response inin mesenteric segments. An enhanced vasoconstriction response was observed MtS group, and the supplementation with Prodefen ® Plus for 4 weeks diminished this vasoconstriction endothelium-denuded mesenteric segments. An enhanced vasoconstriction response was observed response (Figure 5b). We next inhibited the NO synthesis in MtS group, and the supplementation with Prodefen® Plus with an unspecific NO synthase for 4 weeks diminished (NOS) this inhibitor vasoconstriction response (Figure 5b). We next inhibited the NO synthesis with an unspecificand L-NAME (0.1 mmol/L) and observed a potentiation in the vasoconstriction response in CT NO MtS-SYNB groups, synthase (NOS) while itL-NAME inhibitor exerted no effect (0.1 in arteries mmol/L) from animals and observed from MtS in a potentiation group (Figure 6a–d). the vasoconstriction response in CT and MtS-SYNB groups, while it exerted no effect in arteries from animals from MtS group (Figure 6a–d). Nutrients 2020, 2020, 12, 12, 117 x FOR PEER REVIEW 10 of 11 of 18 19 Figure 6. Effect of preincubation with 0.1 mmol/L L-NAME (non-specific NOS inhibitor) on the Figure 6. Effect of preincubation with 0.1 mmol/L L-NAME (non-specific NOS inhibitor) on the vasoconstrictor response elicited by EFS in mesenteric segments from CT (a), MtS (b) and MtS-SYNB (c) vasoconstrictor response elicited by EFS in mesenteric segments from CT (a), MtS (b) and MtS-SYNB rats. Results (mean ± S.E.M.) are expressed as a % of previous contraction elicited by KCl. Statistical (c) rats. Results (mean ± S.E.M.) are expressed as a % of previous contraction elicited by KCl. Statistical analysis: Paired two-way ANOVA. $ p < 0.05 segments without L-NAME vs. segments with L-NAME. analysis: Paired two-way ANOVA. $ p < 0.05 segments without L-NAME vs. segments with L-NAME. (d) Differences of area under the curve (dAUC) in the absence or presence of 0.1 mmol/L L-NAME. (d) Differences of area under the curve (dAUC) in the absence or presence of 0.1 mmol/L L-NAME. Statistical analysis: One-way ANOVA followed by a Newman–Keuls post-hoc test. * p < 0.05 CT vs. Statistical analysis: One-way ANOVA followed by a Newman–Keuls post-hoc test. * p < 0.05 CT vs. MtS; # p < 0.05 MtS vs. MtS-SYNB. (e) Expression of nNOS in mesenteric segments from CT, MtS and MtS; # p < 0.05 MtS vs. MtS-SYNB. (e) Expression of nNOS in mesenteric segments from CT, MtS and MtS-SYNB rats. The blot is representative of eight separate segments from each group. The graph on MtS-SYNB rats. The blot is representative of eight separate segments from each group. The graph on the right shows densitometric analyses of nNOS expression. Results (mean ± S.E.M.) are expressed as the right shows densitometric analyses of nNOS expression. Results (mean ± S.E.M.) are expressed as the ratio of the signal obtained for nNOS and that obtained for β-actin. the ratio of the signal obtained for nNOS and that obtained for β-actin. 3.3.3. Mechanisms Implicated in Neuronal Nitric Oxide Release 3.3.3. Mechanisms Implicated in Neuronal Nitric Oxide Release Alterations in nNOS expression and/or activity can be the responsible for the differences in NO Alterations release observed.inWe nNOS foundexpression and/or activity that the expression can be of nNOS was the responsibleamong comparable for thegroups differences (Figurein 6e). NO release observed. We found that the expression of nNOS was comparable among PKA, PKC and PI3K/AKT signalling pathways play a crucial role in the activation of nNOS. groups (Figure 6e). PKA, the To analyze PKC and PI3K/AKT involvement of thesignalling PKA pathway,pathways play a crucial we preincubated therole in the activation ofmesenteric endothelium-denuded nNOS. To analyze the segments withinvolvement a PKA inhibitorof H89 the (1PKA µmol/L)pathway, before EFSwe stimulation preincubated the endothelium-denuded and measurement of NO release. mesenteric We observedsegments that H89with a PKA inhibitor diminished EFS-induced H89NO(1 μmol/L) before EFS release similarly stimulation in the CT groupand measurement (60.69 ± 3.45% of of NO release. inhibition) andWe observed that in MtS-SYNB groupH89 diminished (50.19 ± 13.95% EFS-induced of inhibition) NOwhile release similarly ainlower it produced the CT group effect in (60.69 MtS ± 3.45% group of inhibition) (20.48 ± 11.79% ofand in MtS-SYNB inhibition). group We next (50.19 ±the analysed 13.95% of inhibition) involvement of thewhile it produced PKC pathway by a lower using effect a PKC in MtS Calfostin inhibitor group (20.48 ± 11.79% C (0.1 µmol/L),offindig inhibition). We a similar next analysed inhibition the involvement by in EFS-induced of the NO release PKC pathway among by using a PKC the experimental groups inhibitor (% of Calfostin inhibition:C (0.1 CT: μmol/L), findig 76.41 ± 6.53; a similar MtS: 57.33 inhibition by in EFS- ± 22.64; MtS-SYNB: 57.54 ± 12.71). Similar results were found after preincubation with LY394002 (10 µmol/L), a57.33 induced NO release among the experimental groups (% of inhibition: CT: 76.41 ± 6.53; MtS: PI3K± 22.64; MtS-SYNB: inhibitor 57.54 ± CT: (% of inhibition: 12.71). Similar 83.96 ± 8.61;results were±found MtS: 60.51 10.06;after preincubation MtS-SYNB: 64.22 ± with 8.46) LY394002 (10 (Figure 7a–c). μmol/L), a PI3K inhibitor (% of inhibition: CT: 83.96 ± 8.61; MtS: 60.51 ± 10.06; MtS-SYNB: 64.22 ± 8.46) (Figure 7a–c). Nutrients 2020, 12, x FOR PEER REVIEW 12 of 19 Nutrients 2020, 12, 117 11 of 18 Nutrients 2020, 12, x FOR PEER REVIEW 12 of 19 Figure 7. Mesenteric segments from CT (a), MtS (b) and MtS-SYNB (c) rats were preincubated with a Figure Figure Mesenteric segments 7. Mesenteric segments from fromCTCT (a),MtS MtS(b)(b)and andMtS-SYNB MtS-SYNB(c) (c)rats ratswere werepreincubated preincubated witha protein7.kinase A –PKA- inhibitor H89 (1(a), μmol/L), a protein kinase C –PKC- inhibitor Calfostinwith C (0.1 aprotein protein kinase A –PKA- inhibitor H89 (1 µmol/L), a protein kinase C –PKC- inhibitor Calfostin C μmol/L)kinase A –PKA- inhibitor H89 or a phosphatidylinositol (1 μmol/L), 3-kinase -PI3K-a protein inhibitorkinase C –PKC- LY294002 inhibitor NO (10 μmol/L). Calfostin C (0.1 release was (0.1 µmol/L)a or μmol/L) a phosphatidylinositol 3-kinase -PI3K- inhibitor LY294002 (10 µmol/L). NO release induced or phosphatidylinositol by EFS. Data are expressed3-kinase -PI3K- as arbitrary inhibitor LY294002 fluorescence (10 μmol/L). units (A.F.U.)/mg NO release tissue. was Statistical was induced by EFS. Data are expressed as arbitrary fluorescence units (A.F.U.)/mg tissue. Statistical induced analysis:by EFS. Data One-way are expressed ANOVA as aarbitrary followed by fluorescence Newman–Keuls units post-hoc test.(A.F.U.)/mg tissue. Statistical $ p < 0.05 conditions without analysis: One-way ANOVA followed by a Newman–Keuls post-hoc test. $ p < 0.05 conditions without analysis: inhibitorOne-way ANOVA vs. conditions withfollowed byinhibitor. respective a Newman–Keuls post-hoc test. $ p < 0.05 conditions without inhibitor vs. conditions with respective inhibitor. inhibitor vs. conditions with respective inhibitor. Thisfinding This findingcoincides coincideswith withthe thePKA PKAactivity, activity, which which waswas significantly significantly reduced reduced in the in the MtSMtS group group as as This finding compared to coincides the other withgroups two the PKA activity, (Figure 8a).which was to Similarly significantly previous reduced PKC findings, in theactivity MtS group and compared to the other two groups (Figure 8a). Similarly to previous findings, PKC activity and AKT as AKTcompared to the expression andother two groups (Figure phosphorylation 8a). Similarly were comparable to previous(Figure findings, PKC activity and expression and phosphorylation were comparable amongamong groupsgroups (Figure 8b,c). 8b,c). AKT expression and phosphorylation were comparable among groups (Figure 8b,c). Figure 8. (a) PKA activity, and (b) PKC activity in mesenteric arteries from CT, MtS and MtS-SYNB rats. Figure 8. Results are(a) PKA activity, represented and (b)density as optical PKC activity in mesenteric (OD) units/µg proteinarteries (mean from CT, MtS ± S.E.M). and MtS-SYNB Statistical analysis: Figure 8. (a) rats. Results PKA activity, are represented and (b) PKC activity in mesenteric arteries from CT, MtS and MtS-SYNB One-way ANOVA followed byasa Newman–Keuls optical density (OD) units/μg post-hoc test. * protein p < 0.05(mean CT vs. ±MtS;S.E. #M). p < Statistical 0.05 MtS rats. vs. Results analysis: MtS-SYNB; are+represented One-way pANOVA < 0.05 CT asvs. optical followed bydensity (OD) a Newman–Keuls MtS-SYNB. (c) units/μg Western blotprotein post-hoc test.(mean analysis *for ± S.E. p