Fructose Intake Impairs the Synergistic Vasomotor Manifestation of Nitric Oxide and Hydrogen Sulﬁde in Rat Aorta

International Journal of Molecular Sciences

Article Fructose Intake Impairs the Synergistic Vasomotor Manifestation of Nitric Oxide and Hydrogen Sulﬁde in Rat Aorta

Andrea Berenyiova *, Samuel Golas, Magdalena Drobna, Martina Cebova and Sona Cacanyiova

Institute of Normal and Pathological Physiology, Centre of Experimental Medicine Slovak Academy of Sciences, 841 04 Bratislava, Slovakia; [email protected] (S.G.); [email protected] (M.D.); [email protected] (M.C.); [email protected] (S.C.) * Correspondence: [email protected]

Abstract: In this study, we evaluated the effect of eight weeks of administration of 10% fructose solution to adult Wistar Kyoto (WKY) rats on systolic blood pressure (SBP), plasma and biometric parameters, vasoactive properties of the thoracic aorta (TA), NO synthase (NOS) activity, and the

expression of enzymes producing NO and H2S. Eight weeks of fructose administration did not affect SBP, glycaemia, or the plasma levels of total cholesterol or low-density and high-density lipoprotein; however, it significantly increased the plasma levels of γ-glutamyl transferase and alanine transaminase. Chronic fructose intake deteriorated endothelium-dependent vasorelaxation (EDVR) and increased the sensitivity of adrenergic receptors to noradrenaline. Acute NOS inhibition evoked a reduction in EDVR that was similar between groups; however, it increased adrenergic contraction more in fructose-fed rats. CSE inhibition decreased EDVR in WKY but not in fructose-fed Citation: Berenyiova, A.; Golas, S.; rats. The application of a H2S scavenger evoked a reduction in the EDVR in WKY rats and normalized Drobna, M.; Cebova, M.; Cacanyiova, the sensitivity of adrenergic receptors in rats treated with fructose. Fructose intake did not change S. Fructose Intake Impairs the NOS activity but reduced the expression of eNOS and CBS in the TA and CSE and CBS in the left Synergistic Vasomotor Manifestation ventricle. Based on our results, we could assume that the impaired vascular function induced by of Nitric Oxide and Hydrogen Sulfide increased fructose intake was probably not directly associated with a decreased production of NO, in Rat Aorta. Int. J. Mol. Sci. 2021, 22, 4749. https://doi.org/10.3390/ijms but rather with impairment of the NO–H2S interaction and its manifestation in vasoactive responses. 22094749 Keywords: fructose; Wistar Kyoto rats; thoracic aorta; nitric oxide; hydrogen sulfide Academic Editor: Marcin Magierowski

Received: 6 April 2021 1. Introduction Accepted: 27 April 2021 Currently, significant changes in nutritional habits present an important factor en- Published: 30 April 2021 croaching into the equilibrium of different signaling pathways in the body. Excessive intake of added sugars, mainly as sugar-sweetened beverages, has been implicated in the Publisher’s Note: MDPI stays neutral development of obesity and metabolic disturbances, increasing the risk of cardiovascular with regard to jurisdictional claims in disease mortality. Two major sweeteners are predominantly used in the food and bev- published maps and institutional affil- erage industry: sucrose, a disaccharide containing 50% fructose and 50% glucose; and iations. high-fructose corn syrup, which mainly consists of 55% fructose and 45% glucose [1,2]. Fructose is a natural, simple sugar found in fruits and honey that is responsible for their sweet taste. Due to its specific metabolism, predominantly in the liver, fructose is more lipogenic than glucose. Long-term application of this saccharide at different concentrations Copyright: © 2021 by the authors. in food or drinking water has been used to induce metabolic disorders in experimental Licensee MDPI, Basel, Switzerland. models [3]. Animals chronically receiving high amounts of fructose developed hypertriglyc- This article is an open access article eridemia, insulin resistance, hyperinsulinemia, and often obesity and hypertension [4–6]. distributed under the terms and Zemancikova and Torok (2014) [7] also reported that eight weeks of fructose administration conditions of the Creative Commons to adult Wistar rats induced metabolic changes associated with an increase in plasma Attribution (CC BY) license (https:// glucose and lipids, enlargement of the liver, significant blood pressure elevation, and creativecommons.org/licenses/by/ reduction in endothelium-derived vasorelaxation. The authors also pointed out the specific 4.0/).

Int. J. Mol. Sci. 2021, 22, 4749. https://doi.org/10.3390/ijms22094749 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 18

in plasma glucose and lipids, enlargement of the liver, significant blood pressure elevation, and reduction in endothelium-derived vasorelaxation. The authors also pointed out the specific role of NO/NOS signaling in these processes, because simultaneous treatment with a NOS inhibitor and fructose did not evoke the abovementioned pathological changes. Literary data show that another gaseous molecule, endogenously synthesized hydro- Int. J. Mol. Sci. 2021, 22, 4749 2 of 17 gen sulfide (H2S), is involved in the same biological processes as NO, including the vasomotor responses of the arterial wall, neurotransmission, control of infectious processes, and insulin release [8,9]. Pan et al. (2014) [10] demonstrated that a high-saccharide envi- role of NO/NOS signaling in these processes, because simultaneous treatment with a NOS ronmentinhibitor andinhibits fructose the did expression not evoke theof abovementionedone of the H2S-producing pathological changes.enzymes, cystathionine-γ- lyaseLiterary (CSE), datain adipocytes. show that another Several gaseous studies molecule, have also endogenously suggested synthesizedthat high glucose hy- intake significantlydrogen sulfide increases (H2S), is involvedthe secretion in the sameof pro-inflammatory biological processes cytokines, as NO, including including the tumor ne- crosisvasomotor factor- responsesα (TNF- ofα the), IL-6, arterial and wall, monocyte neurotransmission, chemoattractant control of protein-1 infectious processes, [11,12], and the in- and insulin release [8,9]. Pan et al. (2014) [10] demonstrated that a high-saccharide environ- volved mechanism is partly related to inhibition of the CSE/H2S system. ment inhibits the expression of one of the H2S-producing enzymes, cystathionine-γ-lyase Based on this, we can assume that the endogenous NO and H2S signaling pathways (CSE), in adipocytes. Several studies have also suggested that high glucose intake signif- couldicantly be increases involved the in secretion the metabolic of pro-inflammatory disorders caused cytokines, by high including saccharide tumor necrosis intake. However, thefactor- dataα (TNF- relatedα), IL-6,to their and vasoregulatory monocyte chemoattractant mechanisms protein-1 in this [11 ,12circumstance], and the involved are still limited. Themechanism main goal is partly of the related present to inhibition study was of the to CSE/H describe2S system. the effect of eight weeks of fructose administrationBased on this, to weadult can Wistar assume thatKyoto the (WKY) endogenous rats NOon the and vasoactive H2S signaling properties pathways of the tho- could be involved in the metabolic disorders caused by high saccharide intake. However, racic aorta, with special emphasis on the participation of NO and H2S signaling pathways. the data related to their vasoregulatory mechanisms in this circumstance are still limited. SuchThe main observations goal of the have present so studyfar not was been to describe made. We the effecthypothesized of eight weeks that ofthe fructose increased intake ofadministration fructose induces to adult metabolic Wistar Kyoto syndrome-like (WKY) rats on thedisorders vasoactive that properties could ofprobably the thoracic modulate the NOaorta, and with H special2S signaling emphasis pathways on the participation and their ofinteraction. NO and H2 S signaling pathways. Such observations have so far not been made. We hypothesized that the increased intake of 2.fructose Results induces metabolic syndrome-like disorders that could probably modulate the NO and H S signaling pathways and their interaction. 2.1. Blood2 Pressure, Cardiac Hypertrophy, Weight Gain, and Fluid and Food Intake 2. ResultsLong-term increased fructose intake had a significant effect on the values of systolic 2.1. Blood Pressure, Cardiac Hypertrophy, Weight Gain, and Fluid and Food Intake blood pressure (SBP) (F(1;219) = 14.25; p = 2.09 × 10−4); however, the statistical analysis did not revealLong-term any increaseddifferences fructose between intake the had experi a significantmental effect groups on the in valuesindividual of systolic weeks (Figure blood pressure (SBP) (F = 14.25; p = 2.09 × 10−4); however, the statistical analysis did 1). Fructose treatment(1;219) had no impact on the parameters of heart trophicity; neither the not reveal any differences between the experimental groups in individual weeks (Figure1 ). ratioFructose of heart treatment weight had noto impactbody weight on the parameters nor the tibial of heart length trophicity; to body neither weight the ratio were changed afterof heart fructose weight treatment to body weight (Table nor 1). the Surprising tibial lengthly, tothe body eight-week weight were fructose changed treatment after did not affectfructose the treatment weight (Table gain1 ).or Surprisingly, adiposity the(expressed eight-week as fructose a ratio treatment of the retroperitoneal did not affect adipose tissuethe weight weight gain to or the adiposity tibia length) (expressed of the as animals a ratio of (Table the retroperitoneal 1). Although adipose the animals tissue receiving theweight fructose to the solution tibia length) had of an the increased animals (Table average1). Although weekly the liquid animals intake, receiving they the had a signifi- fructose solution had an increased average weekly liquid intake, they had a significantly cantlylower weeklylower foodweekly intake food than intake the control than groupthe control (Figure group2a,b). (Figure 2a,b).

Figure 1. Time course of systolic blood pressure in Wistar Kyoto (WKY) and in Wistar Kyoto rates Figuretreated 1. with Time 10% course fructose of solution systolic (WKY blood + FRU) pressure (n = 6). in Data Wistar are expressedKyoto (WKY) as the and mean in± WistarSEM. Kyoto rates treated with 10% fructose solution (WKY + FRU) (n = 6). Data are expressed as the mean ± SEM.

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Int. J. Mol. Sci. 2021, 22, 4749 Table 1. General parameters of the experimental animals. 3 of 17

HW/TL n WG (g) HW/BW (mg/g) RW/TL (mg/mm) (mg/mm) Table 1. General parameters of the experimental animals. WKY 6 78.7 ± 7.91 3.37 ± 0.1 29.7 ± 2.2 96.5 ± 16.9 HW/BW HW/TL RW/TL WKY + FRU 6 n 80.5 ± 8.7 WG (g) 3.49 ± 0.1 31.4 ± 1.1 82.8 ± 14.8 (mg/g) (mg/mm) (mg/mm) WKY—WistarWKY Kyoto rats; 6 WKY + FRU—Wistar 78.7 ± 7.91 Kyoto 3.37 rats± treated0.1 with 29.7 10%± fructose2.2 solution; 96.5 ± 16.9 WG—weight gain; HW/BW—ratio of the heart weight to body weight; HW/TL—ratio of the heart WKY + FRU 6 80.5 ± 8.7 3.49 ± 0.1 31.4 ± 1.1 82.8 ± 14.8 weight to tibia length; RW/TL—ratio of the retroperitoneal adipose tissue weight to tibia length. WKY—Wistar Kyoto rats; WKY + FRU—Wistar Kyoto rats treated with 10% fructose solution; WG—weight Data are expressed as the mean ± SEM. gain; HW/BW—ratio of the heart weight to body weight; HW/TL—ratio of the heart weight to tibia length; RW/TL—ratio of the retroperitoneal adipose tissue weight to tibia length. Data are expressed as the mean ± SEM.

90 22

80 20

70 18 16 60 14 50 12 ** 40 ** 10

30 (g) intake Food Liquid intake (ml) 6 20 4 WKY 10 WKY 2 WKY + FRU WKY + FRU 0 0 0123456789 0123456789 Weeks Weeks

(a) (b)

FigureFigure 2. 2. TimeTime course course of of liquid liquid (a (a) )and and food food (b (b) )intake intake in in Wistar Wistar Kyoto Kyoto (WKY) (WKY) and and in in Wistar Wistar Kyoto Kyoto rats rats treated treated with with 10% 10% fructosefructose solution solution (WKY (WKY + + FRU) FRU) (n (n == 6). 6). Data Data are are expressed expressed as as the the mean mean ±± SEM.SEM. ** **p

2.2.2.2. Plasma Plasma Parameters Parameters TheThe levels levels of of triacylglycerols (TAG), high-densityhigh-density cholesterolcholesterol (HDL),(HDL), alanine alanine amino- ami- notransferasetransferase (ALT), (ALT), gamma gamma glutamyl glutamyl transferase transferase (γ-GT), (γ-GT), cystatin cystatin C (CYS), C (CYS), creatinine creatinine (CRE) (CRE)and glucose and glucose (GLU) (GLU) were determined were determined in plasma in plasma samples samples from truncal from truncal blood. Fructoseblood. Fruc- treat- tosement treatment had no impact had no on impact the TAG, on HDL,the TAG, CYS, HDL, or GLU CYS, levels, or GLU but signiﬁcantly levels, but significantly increased the increasedALT and γthe-GT ALT levels, and indicating γ-GT levels, liver indicating injury (Table liver2). injury Moreover, (Table there 2). Moreover, was a non-signiﬁcant there was atrend non-significant of CRE level trend increase of CRE (p

Endothelial functions were tested by the application of cumulative doses of acetylcholine (Ach, 10−10 to 10−5 mol/L), which induces vasorelaxation mediated by NO produced in endothelial cells. Chronic fructose intake signiﬁcantly reduced endothelium- dependent vasorelaxation (Figure3a). Cumulative application of exogenous noradrenaline −10 −6 (NA, 10 to 3 × 10 mol/L) induced vasoconstriction in a concentration-dependent man- Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 18

2.3. Vasoactive Responses Endothelial functions were tested by the application of cumulative doses of acetylcholine (Ach, 10−10 to 10−5 mol/L), which induces vasorelaxation mediated by NO produced in endothelial cells. Chronic fructose intake significantly reduced endothelium-dependent Int. J. Mol. Sci. 2021, 22, 4749 vasorelaxation (Figure 3a). Cumulative application of exogenous noradrenaline (NA,4 of 10 17−10 to 3 × 10−6 mol/L) induced vasoconstriction in a concentration-dependent manner by activating adrenergic receptors. Although the absolute force of contractile responses was not neraffected by activating by the adrenergictreatment (Figure receptors. 3b), Although it significantly the absolute increased force the of contractilesensitivity responses of the ad- wasrenergic not affected receptors by the(F(1;258) treatment = 11.53; (Figure p = 7.83 b),× 10 it− signiﬁcantly4) (Figure 3c). increased The vasorelaxant the sensitivity effect of of the the exogenous NO donor sodium nitroprusside (NS,−4 10−10 to 10−5 mol/L) was not affected by adrenergic receptors (F(1;258) = 11.53; p = 7.8 × 10 ) (Figure3c). The vasorelaxant effect of thechronic exogenous fructose NO intake donor (Figure sodium 3d). nitroprusside (NS, 10−10 to 10−5 mol/L) was not affected by chronic fructose intake (Figure3d).

80 *** Relaxation (%)

100 Control 120 Fru

10 3x10 9 3x9 8 3x8 7 3x7 6 3x6 5 Acetylcholine (-log mol/L)

(a) (b)

Control 100 0 Fru

20 80

40 60 * 60 40 80 Relaxation (%) Contraction (%) Contraction 20 100 Control 0 120 Fru

10 3x10 9 3x9 8 3x8 7 3x7 6 3x6 10 3x10 9 3x9 8 3x8 7 3x7 6 3x6 5 Noradrenaline (-log mol/L) Sodium nitroprusside (-log mol/L)

FigureFigure 3. 3. TheThe effect of chronic chronic increased increased fructose fructose intake intake on on the the endothelium-derived endothelium-derived vasorelaxation vasorelaxation (a),( aadrenergic), adrenergic con- contractiontraction (b (),b sensitivity), sensitivity of ofthe the adrenergic adrenergic receptors receptors (c), (c and), and vasorelaxant vasorelaxant effect effect of ofsodium sodium nitroprusside nitroprusside (d). (d Control—iso-). Control— isolatedlated thoracic thoracic aorta aorta of of Wistar Wistar Kyoto Kyoto rats; rats; Fru—isolated Fru—isolated thoracic thoracic aorta aorta of Wistar of Wistar Kyoto Kyoto rats rats treated treated with with 10% 10% fructose fructose solution (n = 6). Data are expressed as the mean ± SEM. * p < 0.05; *** p < 0.001 vs. Control. solution (n = 6). Data are expressed as the mean ± SEM. * p < 0.05; *** p < 0.001 vs. Control.

ToTo evaluate evaluate the the participation participation of of endogenous endogenous NO NO in in the the basic basic vasoactive vasoactive responses, responses, wewe incubated incubated the the aorticaortic ringsrings withwith NG-nitro-L-arginine-nitro-L-arginine methylester methylester (LN; (LN; 10 10−4− mol/L).4 mol/L). LN LNincubation incubation significantly significantly inhibited inhibited endothelium-dependent endothelium-dependent vasorelaxation vasorelaxation to tothe the same same de- degree in both experimental groups (Figure4a). On the other hand, acute LN treatment increased the contractile response to exogenous NA, and this increase was significantly higher after fructose treatment than in control animals. Not only the treatment (F(1;218) = 34.62; p = 2.08 × 10−8), but also the applied inhibitor (F = 90.62; p = 0) had a significant (1;218) effect on these responses; moreover, a synergistic interaction between the treatment and −4 the inhibitor (F(1;218) = 14.71; p = 1.77 × 10 ) was proven (Figure4b). The sensitivity of −5 the adrenergic receptors was affected by the treatment (F (1;218) = 18.46; p = 2.83 × 10 ) Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 18

gree in both experimental groups (Figure 4a). On the other hand, acute LN treatment increased the contractile response to exogenous NA, and this increase was significantly higher after fructose treatment than in control animals. Not only the treatment (F(1;218) = 34.62; p = 2.08 × 10−8), but also the applied inhibitor (F(1;218) = 90.62; p = 0) had a significant effect on these responses; moreover, a synergistic interaction between the treatment and Int. J. Mol. Sci. 2021, 22, 4749 the inhibitor (F(1;218) = 14.71; p = 1.77 × 10−4) was proven (Figure 4b). The sensitivity 5of of the 17 adrenergic receptors was affected by the treatment (F (1;218) = 18.46; p = 2.83 × 10−5) and the inhibitor (F (1;218) = 4.15; p = 0.04), which were generally caused by the increased sensitivity in fructose-fed rats (Figure 4c). and the inhibitor (F (1;218) = 4.15; p = 0.04), which were generally caused by the increased sensitivity in fructose-fed rats (Figure 4c).

(a)

(b) (c)

FigureFigure 4. 4.The The participation participation of of the the endogenous endogenous NO NO in in the the endothelium-derived endothelium-derived vasorelaxation vasorelaxation ( a(),a), adrenergic adrenergic contraction contraction G (b(b),), and and sensitivity sensitivity ofof thethe adrenergicadrenergic receptorsreceptors ((cc).). Control Control LN LN−−: isolated: isolated thoracic thoracic aorta aorta of ofWistar Wistar Kyoto Kyoto rats rats before before N - −4 Nnitro-L-arginineG-nitro-L-arginine methylester methylester (LN (LN 10 10 mol/L)−4 mol/L) incubation; incubation; Control Control LN+: LN+: isolated isolated thoracic thoracic aorta aorta of Wistar of Wistar Kyoto Kyoto rats ratsafter LN incubation; Fru LN−: isolated thoracic aorta of Wistar Kyoto rats treated with 10% fructose solution before LN incuba- after LN incubation; Fru LN−: isolated thoracic aorta of Wistar Kyoto rats treated with 10% fructose solution before LN tion; Fru LN+: isolated thoracic aorta of Wistar Kyoto rats treated with 10% fructose solution before LN incubation (n = 6). incubation; Fru LN+: isolated thoracic aorta of Wistar Kyoto rats treated with 10% fructose solution before LN incubation Data are expressed as the mean ± SEM. *** p < 0.001 vs. Control LN-; +++ p < 0.001 vs. Fru LN-; ### p < 0.001 vs. Control (nLN+.= 6). Data are expressed as the mean ± SEM. *** p < 0.001 vs. Control LN-; +++ p < 0.001 vs. Fru LN-; ### p < 0.001 vs. Control LN+.

L-Cysteine (Cys; 5 × 10−3 mol/L), a precursor of endogenous H2S production, pyri- L-Cysteine (Cys; 5 × 10−3 mol/L), a precursor of endogenous H S production, pyridoxal 5’-phosphate (PLP; 2 × 10−3 mol/L), a cofactor of cystathionine-2 γ-lyase (CSE), and doxal 5’-phosphate (PLP; 2 × 10−3 mol/L), a cofactor of cystathionine-γ-lyase (CSE), and cystathionine β-synthase (CBS) were added to the organ bath. Cys and PLP treatment did cystathionine β-synthase (CBS) were added to the organ bath. Cys and PLP treatment did not induce any changes in endothelium-dependent vasorelaxation, but had a significant not induce any changes in endothelium-dependent vasorelaxation, but had a signiﬁcant ef- effect on the maximal force of the adrenergic contraction in the control group (F(1;240) = fect on the maximal force of the adrenergic contraction in the control group (F(1;240) = 10.54; p = 0.001; Figure5a–c). Acute treatment with DL-propargylglycine (PPG; 10−2 mol/L) signiﬁcantly decreased the vasorelaxant response to Ach in untreated rats and did not affect

the contractile response to NA (Figure6a–c), whereby the treatment–inhibitor interaction revealed no effect on these responses. The incubation with a H2S scavenger, namely, bismuth (III) subsalicylate (BSC; 10−6 mol/L), signiﬁcantly reduced endothelium-dependent vasorelaxation in control rats and increased the vasorelaxation in rats receiving fructose solution (Figure7a); moreover, the treatment–concentration–scavenger interaction had a Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 18

10.54; p = 0.001; Figure 5a–c). Acute treatment with DL-propargylglycine (PPG; 10−2 mol/L) significantly decreased the vasorelaxant response to Ach in untreated rats and did not affect the contractile response to NA (Figure 6a–c), whereby the treatment–inhibitor interaction revealed no effect on these responses. The incubation with a H2S scavenger, namely, bismuth (III) subsalicylate (BSC; 10−6 mol/L), significantly reduced endothelium-dependent vasorelaxation in control rats and increased the vasorelaxation in rats receiving fruc- Int. J. Mol. Sci. 2021, 22, 4749 tose solution (Figure 7a); moreover, the treatment–concentration–scavenger6 ofinteraction 17 had a significant effect on this response (F(1;202) = 1.97; p = 0.032). The fructose treatment and the scavenger application also affected the adrenergic contractile (F(1;228) = 6.62; p = 0.013;signiﬁcant resp. effectF(1;228) on= 24.88; this response p = 1.37 (F( 1;202)× 10−=6) 1.97; responsesp = 0.032). and The the fructose sensitivity treatment of the and adrenergic the scavenger application also affected the adrenergic contractile (F = 6.62; p = 0.013; resp. (1;228) (1;228) (1;228) −4 receptors (F = 5.32; p = 0.022;−6 resp. F = 12.54; p = 4.99 × 10 ) (Figure 7b,c). F(1;228) = 24.88; p = 1.37 × 10 ) responses and the sensitivity of the adrenergic receptors −4 (F(1;228) = 5.32; p = 0.022; resp. F(1;228) = 12.54; p = 4.99 × 10 ) (Figure7b,c).

(a)

(b) (c)

FigureFigure 5. The 5. effectThe effect of the of theincubation incubation with with a aprec precursorursor and aa cofactor cofactor of of the the endogenous endogenous H2S H synthesis2S synthesis on the on endothelium- the endothelium- derivedderived vasorelaxation vasorelaxation (a), ( aadrenergic), adrenergic contraction contraction ((bb)) and and sensitivity sensitivity of theof the adrenergic adrenergic receptors receptors (c). Control (c). Control Cys/PLP Cys/PLP−: −: isolatedisolated thoracic thoracic aorta aorta of Wistar of Wistar Kyot Kyotoo rats rats before before L-cysteine (Cys;(Cys; 5 5× ×10 10−3−3 mol/L)mol/L) andand pyridoxal pyridoxal 50 -phosphate5′-phosphate (PLP; (PLP; 2 × 10−3 mol/L)2 × 10− incubation;3 mol/L) incubation; Control Control Cys/PLP+: Cys/PLP+: isolated isolated thoracic thoracic aorta aorta of ofWistar Wistar Kyoto Kyoto rats afterafter Cys/PLP Cys/PLP incubation; incubation; Fru Cys/PLPFru− Cys/PLP: isolated− :thoracic isolated thoracicaorta of aorta Wistar of Wistar Kyoto Kyoto rats ratstreated treated with with 10% 10% fructose fructose solution solution beforebefore Cys/PLP Cys/PLP incubation; incubation; Fru Cys/PLP+:Fru Cys/PLP+: isolated thoracic isolated thoracic aorta of aorta Wist ofar Wistar Kyoto Kyoto rats ratstreated treated with with 10% 10% fructose fructose solution before before Cys/PLP Cys/PLP incubation incubation (n = 6). Data(n = are 6). expressed Data are expressed as the mean as the mean± SEM.± *SEM. p < 0.05 * p < vs. 0.05 Control vs. Control Cys/PLP Cys/PLP−. −.

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(a)

(b) (c)

Figure 6. The participationFigure 6. The of the participation cystathionine of the γ cystathionine-lyase-derivedγ-lyase-derived H2S on the endothelium-derived H2S on the endothelium-derived vasorelaxation vasorelaxation (a), adrenergic (a), contraction (b) andadrenergic sensitivity contraction of the (adrenergicb) and sensitivity receptors of the (c adrenergic). Control receptorsPPG−: isolated (c). Control thoracic PPG− aorta: isolated of Wistar thoracic Kyoto aorta rats of Wistar before DL-propargylglycineKyoto (PPG; rats before 10−2 DL-propargylglycinemol/L) incubation; Control (PPG; 10 −PPG+:2 mol/L) isolated incubation; thorac Controlic aorta PPG+: of Wistar isolated Kyot thoracico rats aortaafter ofPPG Wistar incubation; Fru PPGKyoto−: isolated rats after thoracic PPG incubation; aorta of Wistar Fru PPG Kyoto−: isolated rats thoracictreated aortawith of10% Wistar fructose Kyoto solution rats treated before with 10%PPG fructose incubation; solution Fru PPG+: isolated thoracicbefore PPG aorta incubation; of Wistar Fru Kyoto PPG+: rats isolated treated thoracic with aorta 10% offructose Wistar Kyotosolution rats before treated PPG with 10%incubation fructose ( solutionn = 6). Data before are expressed as thePPG mean incubation ± SEM. *** (n = p 6). < 0.001 Data arevs. expressed Control PPG as the−.mean ± SEM. *** p < 0.001 vs. Control PPG−.

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(a)

(b) (c)

FigureFigure 7. 7.The The effect effect of of a a H H2S2S scavenger scavenger on on the the endothelium-derived endothelium-derived vasorelaxation vasorelaxation ( a(),a), adrenergic adrenergic contraction contraction ( b(),b), and and sensitivitysensitivity of of the the adrenergic adrenergic receptors receptors (c ().c). Control Control BSC BSC−−:: isolated isolated thoracic thoracic aorta aorta of of Wistar Wistar Kyoto Kyoto rats rats before before bismuth bismuth (III) (III) −6 subsalicylatesubsalicylate (BSC; (BSC; 10 10−6 mol/L) mol/L) incubation; ControlControl BSC+:BSC+: isolated thoracicthoracic aortaaorta ofof WistarWistarKyoto Kyoto rats rats after after BSC BSC incubation; incubation; Fru BSC−: isolated thoracic aorta of Wistar Kyoto rats treated with 10% fructose solution before BSC incubation; Fru BSC+: Fru BSC−: isolated thoracic aorta of Wistar Kyoto rats treated with 10% fructose solution before BSC incubation; Fru BSC+: isolated thoracic aorta of Wistar Kyoto rats treated with 10% fructose solution before BSC incubation (n = 6). Data are isolated thoracic aorta of Wistar Kyoto rats treated with 10% fructose solution before BSC incubation (n = 6). Data are expressed as the mean ± SEM. *** p < 0.001 vs. Control BSC−; +++ p < 0.001 vs. Fru BSC−. expressed as the mean ± SEM. *** p < 0.001 vs. Control BSC−; +++ p < 0.001 vs. Fru BSC−.

The cumulative application of the H2S donor (Na2S·9H2O; 20, 40, 80, 10, 200, and 400 The cumulative application of the H S donor (Na S·9H O; 20, 40, 80, 10, 200, and μmol/L) on NA-pre-contracted TA rings induced2 a dual2 effect2 in the experimental groups: 400 µmol/L) on NA-pre-contracted TA rings induced a dual effect in the experimental lower concentrations of Na2S induced contraction, whereas higher concentrations evoked groups: lower concentrations of Na2S induced contraction, whereas higher concentrations evokedvasorelaxation vasorelaxation of the arterial of the arterial wall (Figure wall (Figure 8a,b). 8Chronica,b). Chronic increased increased fructose fructose intake intake had no effect on the vasoactive responses induced by the H2S donor. To evaluate the participation had no effect on the vasoactive responses induced by the H2S donor. To evaluate the −4 participationof endogenous of endogenousNO in these NOresponses, in these aortic responses, segments aortic were segments first incubated were first with incubated LN (10 withmol/L). LN (10Acute−4 mol/L). NO deficiency Acute NO significantly deficiency increased significantly the increased relaxant part the relaxant of the dual part vasoac- of the tive response to the H2S donor in control WKY rats, but not in rats treated with fructose dual vasoactive response to the H2S donor in control WKY rats, but not in rats treated with solution (Figure 8a). The KATP channel inhibitor glibenclamide (GLY, 10−4 mol/L)−4 signifi- fructose solution (Figure8a). The K ATP channel inhibitor glibenclamide (GLY, 10 mol/L) cantly reduced the maximum of H2S donor-induced vasorelaxation similarly in both ex- significantly reduced the maximum of H2S donor-induced vasorelaxation similarly in both experimentalperimental groups, groups, demonstrating demonstrating a crucial role ofof hyperpolarizationhyperpolarization inin these these vasoactive vasoactive responsesresponses (Figure (Figure8 b),8b), which which was was unaffected unaffected by by fructose fructose treatment. treatment. Both Both LN LN and and GLY GLY (1;121) (1;122) −12−12 incubationincubation had had a a significant significant (F (F(1;121) = = 5.47;5.47;p p= = 0.02;0.02;F F(1;122) == 59.87; 59.87; pp = 8.38 8.38 ×× 1010 ) effect) effect on onH2 HS 2donor-evokedS donor-evoked vasoactive vasoactive responses. responses.

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(a) (b) (a) (b) FigureFigure 8.8.The The dualdual vasoactivevasoactive effect effect of of H H2S2S donordonor and and the the participation participation of of the the endogenous endogenous NO NO (a ()a and) and K KATPATPchannels channels ( b(b)) in in Figure 8. The dual vasoactive effect of H2S donor and the participation of the endogenous NO (a) and KATP channels (b) in thethe vasoactive vasoactive responses. responses. Control Control LN LN−−:: isolatedisolated thoracicthoracic aortaaorta ofof WistarWistar KyotoKyoto ratsrats beforebefore NNGG-nitro-L-arginine-nitro-L-arginine methylestermethylester the vasoactive responses. Control LN−: isolated thoracic aorta of Wistar Kyoto rats before NG-nitro-L-arginine methylester (LN 10−−44 mol/L) incubation; Control LN+: isolated thoracic aorta of Wistar Kyoto rats after LN incubation; Fru LN−: isolated −4 (LN 10 mol/L) incubation; Control LN+: isolated thoracic aorta of Wistar Kyoto rats after LN incubation; Fru LN−: (LN 10 mol/L) incubation;thoracic Controlaorta of LN+:Wistar isolated Kyoto thoracic rats treated aorta with of Wistar 10% Kyotofructose rats solution after LN before incubation; LN incubation Fru LN−:; isolatedFru LN+: isolated thoracic isolated thoracic aorta of Wistar Kyoto rats treated with 10% fructose solution before LN incubation; Fru LN+: isolated thoracic aorta of Wistaraorta ofKyoto Wistar rats Kyoto treated rats with treated 10% with fructose 10% solutionfructose solutionbefore LN before incubation LN incubation;; Fru LN+: Control isolated GLY thoracic−: isolated thoracic aorta thoracic aorta of Wistar Kyoto rats treated with 10% fructose solution before LN incubation; Control GLY−: isolated thoracic aorta of Wistar Kyotoof Wistarrats treated Kyoto with rats 10% befo frucre glibenclamidetose solution before(GLY, LN10− 4incubation; mol/L) incubation; Control GLYControl−: isolated GLY+: thoracicisolated aortathoracic aorta of Wistar −4 −4 of Wistar Kyoto ratsaortaKyoto befo of rerats Wistar glibenclamide after Kyoto GLY incubation; rats (GLY, before 10 Fru glibenclamide mol/L) GLY− incubation;: isolated (GLY, thoracic 10Controlmol/L) aorta GLY+: of incubation; Wistar isolated Kyoto thoracic Control rats treatedaorta GLY+: of with isolatedWistar 10% thoracicfructose aortasolution of Kyoto rats after GLYWistarbefore incubation; KyotoGLY incubation; rats Fru after GLY GLY− :Fru isolated incubation;GLY+: thoracic isolated Fru aorta GLYthoracic of− :Wistar isolated aorta Kyoto of thoracic Wi ratsstar treated aortaKyoto of rats with Wistar treated 10% Kyoto fructose with rats 10% solution treated fructose with solution 10% fructose before before GLY incubation;solutionGLY incubationFru before GLY+: GLY (isolatedn = incubation; 6). Data thoracic are Fru expressed aorta GLY+: of Wi isolatedasstar the meanKyoto thoracic ± rats SEM. aortatreated * p of< 0.05; Wistarwith *** 10%Kyoto p < fructose 0.001 rats vs. treated solution Control with beforeLN 10%− resp. fructose Control solution GLY−; GLY incubation (nbefore =+++ 6). p Data < GLY 0.001 are incubation vs.expressed Fru GLY (n as=− 6).;the # p Data mean< 0.05 are ± vs. expressedSEM. Control * p < as0.05;LN+. the *** mean p < 0.001± SEM. vs.* Controlp < 0.05; LN ***−p resp.< 0.001 Control vs. Control GLY−; LN − resp. Control +++ p < 0.001 vs. Fru GLY−; # p < 0.05 vs. Control LN+. GLY−; +++ p < 0.001 vs. Fru GLY−;# p < 0.05 vs. Control LN+. 2.4. Total Activity of NO Synthase 2.4. Total Activity of NO Synthase 2.4. TotalThe Activitymeasurement of NO Synthaseof total NOS activity did not reveal any changes in either the left The measurement of total NOS activity did not reveal any changes in either the left ventricleThe measurement(LV) of the heart of totalor the NOS TA activity(Figure 9). did not reveal any changes in either the left ventricle (LV) of the heart or the TA (Figure 9). ventricle (LV) of the heart or the TA (Figure9).

22 22 WKY WKY 20 WKY + Fru 20 WKY + Fru 18 18 16 16 14 14 12 12 10 10 8 8 6 6 NOS activity (pkat/g) NOS activity (pkat/g) 4 4 2 2 0 0 LV TA LV TA

Figure 9. Total activity of NO synthase in different tissues. LV—left ventricle; TA—thoracic aorta;

WKY—WistarFigure 9. Total Kyoto activity rats; of WKYNO synthase + Fru—Wistar in different Kyoto tissues. rats treated LV—left with ventricl 10% fructosee; TA—thoracic solution (aorta;n = 6). Figure 9. Total activity of NO synthase in different tissues. LV—left ventricle; TA—thoracic aorta; DataWKY—Wistar are expressed Kyoto as therats; mean WKY± + SEM.Fru—Wistar Kyoto rats treated with 10% fructose solution (n = WKY—Wistar Kyoto6). rats;Data WKY are expressed + Fru—Wistar as the Kyoto mean rats± SEM. treated with 10% fructose solution (n = 6). Data are expressed2.5. as Expression the mean of± SEM. Enzymes That Produce NO and H2S Our results demonstrated a signiﬁcantly decreased expression of endothelial NO synthase (eNOS) in TA but not in LV (Figure 10a,b). The expression of cystathionine γ-lyase (CSE), which is considered a dominant producer of H2S in the arterial wall, was signiﬁcantly

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2.5. Expression of Enzymes That Produce NO and H2S Int. J. Mol. Sci. 2021, 22, 4749 Our results demonstrated a significantly decreased expression of endothelial10 NO of 17 synthase (eNOS) in TA but not in LV (Figure 10a,b). The expression of cystathionine γ- lyase (CSE), which is considered a dominant producer of H2S in the arterial wall, was significantlyreduced in thereduced LV and in therethe LV was and a there non-significant was a non-significant trend of decreased trend of expression decreased inexpres- the TA 2 sion(p < in 0.07; the FigureTA (p < 10 0.07;c,d). Figure The expression 10c,d). The of expression another enzyme of another synthetizing enzyme synthetizing H2S, cystathionine H S, cystathionineβ-synthase (CBS),β-synthase was (CBS), confirmed was confirmed by our measurement; by our measurement; moreover, moreover, it seems it that seems CBS thatexpression CBS expression was probably was probably higher than higher that than of CSE. that Weof CSE. observed We observed significantly significantly reduced CBSre- ducedexpression CBS expression in both the in TA both and the LV TA (Figure and LV 10e,f). (Figure 10e,f).

Figure 10. The expression of NO- and H2S-producing enzymes in different tissues. eNOS—

endothelial NO-synthase (a,b); CSE—cystathionine γ-lyase (c,d); CBS—cystathionine β-synthase Figure(e,f); LV—left10. The expression ventricle ( aof,c ,NO-e); TA—thoracic and H2S-producing aorta (b enzymes,d,f); WKY—Wistar in different Kyototissues. rats; eNOS—endo- WKY + Fru— thelialWistar NO-synthase Kyoto rats treated (a,b); CSE—cystathionine with 10% fructose solution γ-lyase ((nc,=d); 6). CBS—cystathionine Data are expressed β as-synthase the mean (e±,f);SEM. LV—left ventricle (a,c,e); TA—thoracic aorta (b,d,f); WKY—Wistar Kyoto rats; WKY + Fru— ** p < 0.01 vs. WKY. 3. Discussion Metabolic syndrome is characterized by the coexistence of different severe abnor- malities, including hypertension, abdominal obesity, hyperlipidemia, and disorders in insulin signaling. Several studies have conﬁrmed that high fructose intake could alter Int. J. Mol. Sci. 2021, 22, 4749 11 of 17

pathologic processes in animal models as well as in humans, which triggered alterations coupled with metabolic syndrome [13,14]. However, according to other available studies, the question of a direct association of high fructose consumption with an increase in blood pressure represents a controversial issue. Chronic fructose administration significantly elevated SBP in normotensive conditions [1,7,15]; on the other hand, some authors found no impact of this treatment on blood pressure [16,17]. In our study, although we revealed a significant effect of the eight-week fructose administration on the values of systolic blood pressure, there were no differences between the experimental groups in individual weeks (Figure1). In agreement with these results, we did not demonstrate any changes in heart trophicity. Moreover, we did not confirm obesity in rats treated with fructose (Table1). This result could be influenced by a change in the food and liquid intake in the experimental groups. Although the rats receiving fructose had a higher liquid intake, their food intake was significantly lower than that of the control rats (Figure2a,b). Similar results were demonstrated by Miranda et al. (2019) [3], who recorded increased liquid intake and energy intake but decreased food consumption throughout a 12-week-long period of 7% fructose solution treatment without any significant changes in the final body weight of normotensive Wistar rats. It has also been shown that fructose has a unique metabolism, occurring predominantly in the liver, and its long-term consumption is associated with hypertriglyceridemia [2]. In our experiments, the plasma level of triacylglycerols was not changed in fructose-treated animals; the levels of alanine aminotransferase and γ-glutamyl transferase were significantly increased, and there was a trend of increased levels of creatinine in this group, indicating damage to hepatocytes, liver disease, and kidney injury. According to our results, it seems that the eight-week-long fructose administration did not trigger disorders coupled with metabolic syndrome. We assume that these discrepancies are related to the different amounts of fructose consumed, the lengths of the treatment, and the ages and strains of the experimental animals. The experimental animals in this study were treated with 10% fructose solution, which represents a relatively low concentration of this saccharide; however, Miranda et al. (2019) [3] reported that the findings of studies using the highest fructose concentrations may not be compatible with human consumption. White et al. (2013) [18] also demonstrated that experimental rats tolerate high levels of fructose (≥60%), but that gastric stress can appear in humans even at low fructose doses (approximately 10%). In our study, we confirmed that the long-lasting increase in fructose treatment did not affect the maximal force of adrenergic contraction but increased the sensitivity of adrenergic receptors to exogenous noradrenaline and significantly reduced endothelium-dependent vasorelaxation, although did not alter relaxation to NO donors (Figure3a–d). Similar findings were observed by Zemancikova et al. (2014) [7], who reported an unchanged adrenergic contraction but decreased acetylcholine-induced vasorelaxation in the thoracic aorta of normotensive Wistar rats drinking 10% fructose solution. Moreover, they pointed out that fructose did not induce cardiovascular alterations in rats without a functional NO system in the state of NO deficiency (induced by NG-nitro-L-arginine methylester (LN) administration). According to these findings, they concluded that impairment of the NO system may be responsible for the adverse effects induced by high fructose intake. However, other studies did not report attenuated acetylcholine-induced relaxation after fructose treatment. Two-week-long administration of 20% fructose solution to Sprague Dawley rats had no impact on the contraction response to noradrenaline or the relaxant responses mediated by NO in the thoracic aorta [15]. Similarly, the response of the mesenteric vasculature to acetylcholine was unchanged in fructose-fed Wistar rats [2]. Moreover, Lozano et al. (2016) [19] demonstrated that NO-mediated relaxation was not affected by either a high-fructose or high-fat diet, or even simultaneous treatment, in the mesenteric arteries of normotensive rats. Despite the reduced NO-dependent vasorelaxation, our results demonstrate unchanged total NOS activity measured in the left heart ventricle (LV) and thoracic aorta (TA) of fructose-receiving rats. On the other hand, the protein expression of the eNOS isoform was reduced in the TA but comparable between the Int. J. Mol. Sci. 2021, 22, 4749 12 of 17

experimental groups in the LV (Figures9 and 10). Apart from eNOS, there are two other important isoforms of NOS in the cardiovascular system: the neuronal (nNOS) and inducible (iNOS) forms. The expression of all three NOS isoforms (eNOS, nNOS, and iNOS) was demonstrated not only in the intima, but also in the media of the TA and mesenteric and pulmonary arteries [20]. Moreover, it has been reported that nNOS and iNOS isoforms are able to produce vasoactively relevant NO to compensate for the downregulated eNOS/NO pathway [21–23]. Thus, the reduced eNOS expression in the TA could be compensated for by some of the other NOS isoforms to maintain the total activity of NOS. To evaluate the participation of the endogenous NO/NOS system in the regulation of vascular tone, we incubated isolated aortic rings with the non-specific NOS inhibitor LN. Acute LN treatment increased the contractile response to exogenous NA significantly more in fructose-fed rats compared to untreated rats (Figure4b,c). On the other hand, we observed significantly inhibited endothelium-dependent vasorelaxation as a uniform vascular response to LN incubation. Both experimental groups had the same degree of inhibition, indicating a similar participation of acetylcholine-induced endogenous NO in the relaxation of the TA (Figure4a). Based on these findings, it seems that fructose intake had a different impact on the participation of basic (independent of the receptor) and receptor-dependent NO production. While the receptor-independent NO component is able to significantly attenuate adrenergic contraction to the normal level, the inhibition of vasorelaxation mediated by receptor-dependent NO after fructose administration is probably not related to the deterioration of endothelial function, because at the level of NOS activity and the functional vasomotor responses, our results did not show any differences. Our previous studies showed an obvious interaction between two important vasoregulatory molecules, NO and H2S, and their pathways and suggested the existence of a coupled nitroso–sulfide signaling pathway [24,25]. Herein, we confirmed that the heterogeneity of specific nitroso–sulfide vasoactive signaling exists depending on the occurrence of different pathological stages, e.g., hypertension or increased plasma glucose levels [26]. Recently, in a non-obese rat model of metabolic syndrome (HTG rats), we confirmed a significantly higher expression of CSE in the arterial wall of the TA compared to control rats, and that the endogenous H2S participated in the inhibition of NO-mediated vasorelaxation [27]. Thus, based on these results, it seems that the presence of inherent metabolic disturbances could significantly affect nitroso and sulfide signaling and their mutual interaction. The aortic tissue has been shown to have much higher H2S levels than other vascular tissues [28]; therefore, in the next part of our study, we focused on the question of whether fructose intake was able to modulate the participation of the nitroso–sulfide signaling pathway in the vasoregulation of the TA. We found that the incubation of aortic rings with L-cysteine, 0 a precursor of endogenous H2S production, and pyridoxal 5 -phosphate, a cofactor of CSE and CBS, increased the maximal force of NA-induced contraction in the control group but had no impact on the vasorelaxation of the TA (Figure5). However, a H 2S scavenger, bismuth (III) subsalicylate (BSC), restored the increased sensitivity of adrenergic receptors in fructose-fed rats to a level similar to that in control rats. In addition, BSC (similarly to propargylglycine, a CSE inhibitor) reduced endothelium-dependent vasorelaxation in control rats (Figures6 and7). The elimination of H 2S by using a scavenger in the control group reduced endothelium-dependent vasorelaxation in the same manner as chronic fructose intake. Therefore, the results could indicate that the observed deterioration of endothelial function in fructose-fed rats was probably associated with the absence of a synergistic in- volvement of endogenous H2S in NO-mediated vasorelaxation. Moreover, H2S itself could probably participate in endothelial dysfunction in these rats because BSC significantly increased the vasorelaxant response in rats receiving fructose solution. The synergistic effect of NO and H2S has been suggested by several studies. Coletta et al. (2012) [29] also showed that NO and H2S are mutually required for the physiological control of vascular function. The authors confirmed that pre-treatment with a low concentration of NaHS (30 µmol/L, 15 min) potentiated the vasorelaxant response of the TA to acetylcholine and to the NO donor, which was accompanied by significantly increased cGMP levels. Chen Int. J. Mol. Sci. 2021, 22, 4749 13 of 17

et al. (2014) [30] demonstrated that the application of a H2S donor increases the expression of eNOS and upregulates eNOS activity due to the phosphorylation of its serine residue. Moreover, the determination of the expression of CSE and CBS in our study also confirmed a significant effect of high fructose intake on endogenous H2S synthesis. In the LV, the expression of both CSE and CBS was reduced after fructose treatment, while in the TA, the expression of CBS was found to be significantly lower and there was a non-significant trend of the decreased CSE expression in the TA. Interestingly, it seems that there was a higher expression of CBS than in CSE in the aortic arterial wall (Figure 10). We previously found similar results in human intrarenal arteries, where we did not record gene expression of CSE in the arterial wall, but the mRNA of CBS was detectable [26]. This finding could explain the evidence that the acute inhibition of CSE revealed weaker vasoactive effects than the H2S scavenger, and BSC could eliminate H2S also produced by CSE-independent sources, including CBS. According to our results, it seems that not only CSE, which is considered a dominant H2S producer in the cardiovascular system, but also CBS significantly contributes to the vasoactive manifestation of endogenous H2S. Recent studies suggested H2S, through application of its donors or as an added substance, as a novel potential pharmacological tool in different pathological circum- stances [31–34]. In our previous papers, we showed that in both renal arteries of spontaneously hypertensive rats and internal lobar arteries of hypertensive patients, the simultaneous application of NO and H2S donors evoked a specific vasoactive effect (enhanced maximum relaxation and changed dynamics, respectively), thus demonstrating the impor- tance of H2S–NO interactions [35]. Application of a H2S donor predominantly induces a biphasic vasoactive response [36,37], which was also confirmed by our results (Figure8a,b). We did not record a significant effect of increased fructose intake on this response, and in both groups, Na2S-induced relaxation was primarily associated with hyperpolarization via the activation of KATP channels, as suggested by Zhao et al. (2011) [38]. According to our previous results, we suggested that during acute NO deficiency, H2S donors are able to exert enhanced vasorelaxation to maintain the vasorelaxant capacity of the arterial wall. Moreover, the response was strengthened in a state of arterial hypertension, which we considered a reserve mechanism of the TA to compensate for the pathological processes [36,37]. We also showed that the higher level of glucose in the plasma of patients modified the vasoactive response of the products of the nitroso–sulfide signaling pathway [26]. Similarly, in this study, fructose treatment changed the participation of the NO system in the vasoactive effect of the H2S donor. Fructose intake diminished the increased vasorelaxant effect of H2S donors observed during acute NO deficiency. Based on our results, it seems that a modification of saccharide metabolism (increased glycaemia or increased fructose intake) could significantly alter the interaction between the H2S donor and (endogenous) NO. To the best of our knowledge, findings emphasizing the crucial role of the mutual interaction between the nitroso and sulfide signaling pathways in fructose-related pathologies have not previously been reported.

4. Materials and Methods 4.1. Guide for the Use and Care of Laboratory Animals Procedures were performed in accordance with the guidelines of the Institute of Normal and Pathological Physiology, Centre Experimental Medicine Slovak Academy of Sciences (INPP CEM SAS), and were approved by the State Veterinary and Food Admin- istration of the Slovak Republic and by an ethics committee according to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientiﬁc Purposes, Directive 2010/63/EU of the European Parliament(08/01/2019). All rats were housed under a 12 h light–12 h dark cycle at a constant humidity (45–65%) and temperature (20–22 ◦C). Animals were provided INPP CEM SAS veterinary care. Twelve- week-old male Wistar Kyoto (WKY) rats were used in this study (n = 12). The animals were randomly divided into two groups of six animals each: control WKY receiving tap water, Int. J. Mol. Sci. 2021, 22, 4749 14 of 17

and WKY receiving eight weeks of 10% fructose solution in their drinking water. Both groups had free access to identical standard laboratory rat chow.

4.2. Basic Cardiovascular and Biometric Parameters The liquid and food intakes were monitored daily. During the 8 weeks of the exper- iment, systolic blood pressure (SBP) was measured by a non-invasive plethysmography method once a week. To follow the effect of fructose intake on body weight (BW), we recorded it weekly, and then the ﬁnal BW was determined before decapitation after brief anaesthetization with CO2. The weight of the heart (HW) and retroperitoneal adipose tissue, and the length of the tibia, were recorded for the calculation of biometric parameters such as cardiac and renal hypertrophy and adiposity.

4.3. Collection of Plasma Samples Plasma samples were collected just after decapitation and frozen in aliquots for bio- chemical determinations. The basic levels of triacylglycerols (TAG), high-density lipoprotein cholesterol (HDL), alanine aminotransferase (ALT), gamma glutamyl transferase (γ- GT), cystatin C (CYS), creatinine (CRE), and glucose (GLU) were commercially determined at the Laboklin GMBH. The values of parameters represented pre-prandial concentrations.

4.4. Functional Study Although the TA is a large conduit artery, to avoid impairment of the endothelium, the TA was carefully cleaned of connective tissue and cut into 5 mm-long rings using a binocular microscope. The rings were vertically ﬁxed between 2 stainless wire triangles and immersed in a 20 ml incubation organ bath with oxygenated (95% O2; 5% CO2) Krebs solution (118 mmol/L NaCl; 5 mmol/L KCl; 25 mmol/L NaHCO3; 1.2 mmol/L MgSO4.7H2O; 1.2 mmol/L KH2PO4; 2.5 mmol/L CaCl2; 11 mmol/L glucose; 0.032 mmol/L CaNa2EDTA) kept at 37 ◦C. The upper triangles were connected to sensors of isometric tension (FSG-01), and the changes in this tension were registered by AD converter Advanced Kymograph software (all from MDE, Budapest, Hungary). A resting tension of 1 g was applied to each ring and maintained throughout a 45–60 min equilibration period.

Experimental Protocol Briefly, concentration-dependent contractile responses were induced by increasing doses of noradrenaline (NA; 10−10 to3 × 10−5 mol/L) in a cumulative manner. Contrac- tions were expressed as developed tension (g) or as a percentage of the maximum tissue responses to the agonist (demonstrating the sensitivity of adrenergic receptors). The relaxant responses were followed on rings pre-contracted with noradrenaline, and after the achievement of a stable plateau, the preparations were exposed to cumulative doses of acetylcholine (Ach; 10−10 to 3 × 10−5 mol/L) and sodium nitroprusside (10−10 to 3 × 10−5 mol/L). The rate of relaxation was expressed as a percentage of the noradrenaline-induced contraction. We evaluated the effect of the non-specific inhibitor of NOS, NG-nitro-L- arginine methylester (LN; 10−4 mol/L); the specific CSE inhibitor, DL-propargylglycine −2 −6 (PPG; 10 mol/L); the H2S scavenger, bismuth(III) subsalicylate (BSC; 10 mol/L); and pyridoxal 50-phosphate hydrate (PLP; 2 × 10−3 mol/L) and L-cysteine (L-Cys; 5 × 10−3 mol/L) as precursors of H2S production on NA-induced contraction and Ach-induced relaxation. All drugs were acutely incubated for 20 min in the organ bath. + Na2S•9H2O was used as a H2S donor that dissociates in water solution to Na and 2− + − S , which reacts with H to yield HS and H2S. We use the term Na2S to encompass the − 2− total mixture of H2S, HS and S . Direct vasoactive effects of Na2S were observed on −6 NA-pre-contracted (10 mol/L) rings by the administration of increasing doses of Na2S (20, 40, 80, 100, 200, 400 µmol/L). The participation of the endogenous NO system in the vasomotor responses of TA was followed before and 20 minutes after pre-treatment with a non-specific inhibitor of NOS, NG-nitro-L-arginine methyl ester (L-NAME; 10−4 mol/L), to block basal and receptor-induced endogenous NO production. Moreover, glibenclamide Int. J. Mol. Sci. 2021, 22, 4749 15 of 17

−4 (GLI; 10 mol/L) was used to demonstrate the participation of KATP channels in these vasoactive responses.

4.5. Measurement of the Total NO Synthase Activity Total NOS activity was determined in crude homogenates of the aorta and left ventricle by measuring the formation of [3H]-L-citrulline from [3H]-L-arginine (ARC, St. Louis, MT, USA), as previously described and slightly modiﬁed by Pechanova et al. [39]. [3H]- L-Citrulline was measured with a Quanta Smart TriCarb Liquid Scintillation Analyzer (Perkin-Elmer, Meriden, CT, USA). NOS activity was expressed as pkat/min per gram of protein.

4.6. Expression of Enzymes Producing NO and H2S Protein expression of eNOS, CBS and CSE was determined in the aorta and left ventricle by Western blot analysis. The samples were probed with primary rabbit polyclonal anti-eNOS (Abcam, Cambridge, UK) and anti-CBS (Proteintech, Manchester, UK) and mouse monoclonal anti-CSE (Proteintech, Manchester, UK) antibodies. Anti-GAPDH and anti-β-actin (Abcam, Cambridge, UK) were used as loading controls for the left ventricle and aorta, respectively. A chemiluminescence ECL system (Bio-Rad, Hercules, CA, USA) was used for band intensity visualization. The ChemiDocTM Touch Imagine System (Image LabTM Touch software, version 5.2, Bio-Rad, Hercules, CA, USA) was used for quantiﬁcation of the bands.

4.7. Statistical Analyses The data are expressed as the mean ± S.E.M. For the statistical evaluation of vasoactive responses between groups, three-way analysis of variance (ANOVA) with the Bonferroni post hoc test was used. To evaluate general cardiovascular and plasma parameters, including NO synthase activity and eNOS, CSE, and CBS expression, one-way ANOVA was used with the Bonferroni post hoc test. Differences between means were considered signiﬁcant at p < 0.05.

5. Conclusions In summary, the present results demonstrated a special interaction between the nitroso and sulﬁde signaling pathways in fructose-fed normotensive rats. Although chronic increased fructose intake did not initiate changes characterizing metabolic syndrome, it impaired endothelium-dependent vasorelaxation, predicting possible cardiovascular complications. Moreover, based on our results, we could assume that this reduction in vasorelaxation in the thoracic aorta was probably not directly associated with the decreased production of NO, but rather with impairment of the NO–H2S interaction. We conﬁrmed that fructose altered the vasomotor manifestation of this interaction at least at two different levels: (i) the contribution of endogenous H2S to NO-mediated vasorelaxation; and (ii) the contribution of endogenous NO to the vasoactive effect of the H2S donor.

Author Contributions: Conceptualization, A.B. and S.C.; methodology, S.C.; validation, S.C. and M.C.; formal analysis, M.D.; investigation, S.G., A.B. and M.C.; resources, A.B.; data curation, M.D.; writing—original draft preparation, A.B.; writing—review and editing, S.C.; visualization, A.B.; supervision, S.C.; project administration, A.B.; funding acquisition, A.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Scientiﬁc Grant Agency of The Ministry of Education, Science, Research, and Sport of the Slovak Republic: VEGA 2/0111/19, 2/0132/20, 2/0103/18. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Ethics Committee of Institute of Normal and Pathological Physiology of Centre of Experimental Medicine SAS. Informed Consent Statement: Not applicable. Int. J. Mol. Sci. 2021, 22, 4749 16 of 17

Data Availability Statement: All data arising from this study are contained within the article and any additional data sharing will be considered by the first author upon request. Acknowledgments: We thank Marian Grman for his help in proper preparation of the stock solution of Na2S•9H2O. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Shaligram, S.; Sanguesa, G.; Akther, F.; Alegret, M.; Laguna, J.C.; Rahimian, R. Differential effects of high consumption of fructose or glucose on mesenteric arterial function in female rats. J. Nutr. Biochem. 2018, 57, 136–144. [CrossRef] 2. Sangüesa, G.; Shaligram, S.; Akthur, F.; Roglans, N.; Laguna, J.C.; Rahimian, R.; Alegret, M. Type of supplemented simple sugar, not merely calorie intake, determines adverse effects on metabolism and aortic function in female rats. Am. J. Physiol. Heart Circ. Physiol. 2017, 312, H289–H304. [CrossRef] 3. Miranda, C.A.; Schönholzer, T.E.; Klöppel, E.; Sinzato, Y.K.; Volpato, G.T.; Damasceno, D.C.; Campos, K.E. Repercussions of low fructose-drinking water in male rats. An. Acad. Bras. Ciências 2019, 91, e20170705. [CrossRef][PubMed] 4. Hannou, S.A.; Haslam, D.E.; McKeown, N.M.; Herman, M.A. Fructose metabolism and metabolic disease. J. Clin. Invest. 2018, 28, 545–555. [CrossRef][PubMed] 5. Hernández-Díazcouder, A.; Romero-Nava, R.; Carbó, R.; Sánchez-Lozada, L.G.; Sánchez-Muñoz, F. High Fructose Intake and Adipogenesis. Int. J. Mol. Sci. 2019, 2, 2787. [CrossRef][PubMed] 6. Abdelrahman, A.M.; Suleimani, Y.M.A.; Ashique, M.; Manoj, P.; Ali, B.H. Effect of infliximab and tocilizumab on fructose-induced hyperinsulinemia and hypertension in rats. Biomed. Pharmacother 2018, 105, 182–186. [CrossRef][PubMed] 7. Zemancikova, A.; Torok, J. Effect of perivascular adipose tissue on arterial adrenergic contractions in normotensive and hypertensive rats with high fructose intake. Physiol. Res. 2017, 66, S537–S544. [CrossRef][PubMed] 8. Szabo, C. Hydrogen sulfide, an enhancer of vascular nitric oxide signaling: Mechanisms and implications. Am. J. Physiol. Cell Physiol. 2017, 312, C3–C15. [CrossRef][PubMed] 9. Gheibi, S.; Samsonov, A.P.; Gheibi, S.; Vazquez, A.B.; Kashfi, K. Regulation of carbohydrate metabolism by nitric oxide and hydrogen sulfide: Implications in diabetes. Biochem. Pharmacol. 2020, 176, 113819. [CrossRef] 10. Pan, Y.; Kong, L.D. High fructose diet-induced metabolic syndrome: Pathophysiological mechanism and treatment by traditional Chinese medicine. Pharmacol. Res. 2018, 130, 438–450. [CrossRef] 11. Grosick, R.; Alvarado-Vazquez, P.A.; Messersmith, A.R.; Romero-Sandoval, E.A. High glucose induces a priming effect in macrophages and exacerbates the production of pro-inflammatory cytokines after a challenge. J. Pain Res. 2018, 11, 1769–1778. [CrossRef][PubMed] 12. Briones, L.; Andrews, M.; Pizarro, F.; Arredondo-Olguin, M. Expression of genes associated with inflammation and iron metabolism in 3T3-L1 cells induced with macrophages-conditioned medium, glucose and iron. Biometals 2018, 31, 595–604. [CrossRef][PubMed] 13. Basciano, H.; Federico, L.; Adeli, K. Fructose, insulin resistance, and metabolic dyslipidemia. Nutr. Metab. 2005, 2, 5. [CrossRef] [PubMed] 14. Evans, R.A.; Frese, M.; Romero, J.; Cunningham, J.H.; Mills, K.E. Chronic fructose substitution for glucose or sucrose in food or beverages has little effect on fasting blood glucose, insulin, or triglycerides: A systematic review and meta-analysis. Am. J. Clin. Nutr. 2017, 106, 519–529. [CrossRef][PubMed] 15. Yoon, S.; Lee, E.; Kim, M.; Kim, I. Acute Exposure to Fructose Impairs Endothelium-Dependent Relaxation via Oxidative Stress in Isolated Rat Aortic Rings. Vasc. Res. 2020, 57, 213–222. [CrossRef] 16. Sousa, G.J.; Oliveira, P.W.C.; Nogueira, B.V.; Melo, A.F., Jr.; Faria, T.O.; Meira, E.F.; Mill, J.G.; Bissoli, N.S.; Baldo, M.P. Fructose intake exacerbates the contractile response elicited by norepinephrine in mesenteric vascular bed of rats via increased endothelial prostanoids. J. Nutr. Biochem. 2017, 48, 21–28. [CrossRef] 17. Rehakova, R.; Klimentova, J.; Cebova, M.; Barta, A.; Matuskova, Z.; Labas, P.; Pechanova, O. Effect of deuterium-depleted water on selected cardiometabolic parameters in fructose-treated rats. Physiol. Res. 2016, 65, S401–S407. [CrossRef][PubMed] 18. White, J.S. Challenging the fructose hypothesis: New perspectives on fructose consumption and metabolism. Adv. Nutr. 2013, 4, 246–256. [CrossRef][PubMed] 19. Lozano, I.; Van der Werf, R.; Bietiger, W.; Seyfritz, E.; Peronet, C.; Pinget, M.; Jeandidier, N.; Maillard, E.; Marchioni, E.; Sigrist, S.; et al. High-fructose and high-fat diet-induced disorders in rats: Impact on diabetes risk, hepatic and vascular complications. Nutr. Metab. 2016, 13, 15. [CrossRef][PubMed] 20. Buchwalow, I.B.; Cacanyiova, S.; Neumann, J.; Samoilova, V.E.; Boecker, W.; Kristek, F. The role of arterial smooth muscle in vasorelaxation. Biochem. Biophys Res. Commun. 2008, 377, 504–507. [CrossRef] 21. Talukder, M.A.H.; Fujiki, T.; Morikawa, K.; Motoishi, M.; Kubota, H.; Morishita, T.; Tsutsui, M.; Takeshita, A.; Shimokawa, H. Up-regulated neuronal nitric oxide synthase compensates coronary flow response to bradykinin in endothelial nitric oxide synthase-deficient mice. J. Cardiovasc. Pharmacol. 2004, 44, 437–445. [CrossRef][PubMed] Int. J. Mol. Sci. 2021, 22, 4749 17 of 17

22. Biecker, E.; Neef, M.; Sägesser, H.; Shaw, S.; Koshy, A.; Reichen, J. Nitric oxide synthase 1 is partly compensating for nitric oxide synthase 3 deficiency in nitric oxide synthase 3 knock-out mice and is elevated in murine and human cirrhosis. Liver Int. 2004, 24, 345–353. [CrossRef][PubMed] 23. Pechanova, O.; Dobesová, Z.; Cejka, J.; Kunes, J.; Zicha, J. Vasoactive systems in L-NAME hypertension: The role of inducible nitric oxide synthase. J. Hypertens. 2004, 22, 167–173. [CrossRef][PubMed] 24. Berenyiova, A.; Grman, M.; Mijuskovic, A.; Stasko, A.; Misak, A.; Nagy, P.; Ondriasova, E.; Cacanyiova, S.; Brezova, V.; Feelisch, M.; et al. The reaction products of sulfide and S-nitrosoglutathione are potent vasorelaxants. Nitric Oxide 2015, 46, 123–130. [CrossRef] 25. Berenyiova, A.; Grman, M.; Misak, A.; Golas, S.; Cuchorova, J.; Cacanyiova, S. The Possible Role of the Nitroso-Sulfide Signaling Pathway in the Vasomotoric Effect of Garlic Juice. Molecules 2020, 25, 590. [CrossRef][PubMed] 26. Cacanyiova, S.; Krskova, K.; Zorad, S.; Frimmel, K.; Drobna, M.; Valaskova, Z.; Misak, A.; Golas, S.; Breza, J.; Breza, J., Jr.; et al. Arterial Hypertension and Plasma Glucose Modulate the Vasoactive Effects of Nitroso-Sulfide Coupled Signaling in Human Intrarenal Arteries. Molecules 2020, 25, 2886. [CrossRef][PubMed] 27. Cacanyiova, S.; Golas, S.; Zemancikova, A.; Majzunova, M.; Cebova, M.; Malinska, H.; Hüttl, M.; Markova, I.; Berenyiova, A. The Vasoactive Role of Perivascular Adipose Tissue and the Sulfide Signaling Pathway in a Nonobese Model of Metabolic Syndrome. Biomolecules 2021, 11, 108. [CrossRef] 28. Levitt, M.D. Free and acid-labile hydrogen sulfide concentrations in mouse tissues: Anomalously high free hydrogen sulfide in aortic tissue. Antioxid. Redox Signal. 2011, 15, 373–378. [CrossRef] 29. Coletta, C.; Papapetropoulos, A.; Erdelyi, K.; Olah, G.; Módis, K.; Panopoulos, P.; Asimakopoulou, A.; Gerö, D.; Sharina, I.; Martin, E.; et al. Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation. Proc. Natl. Acad. Sci. USA 2012, 109, 9161–9166. [CrossRef] 30. Cheng, Y.; Ndisang, J.F.; Tang, G.; Cao, K.; Wang, R. Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats. Am. J. Physiol. Heart Circ. Physiol. 2004, 287, H2316–H2323. [CrossRef] 31. Powell, C.R.; Dillon, K.M.; Matson, J.B. A review of hydrogen sulfide (H2S) donors: Chemistry and potential therapeutic applications. Biochem. Pharmacol. 2018, 149, 110–123. [CrossRef][PubMed] 32. Lee, Z.W.; Deng, L.W. Role of H2S Donors in Cancer Biology. Handb. Exp. Pharmacol. 2015, 230, 243–265. [CrossRef][PubMed] 33. Sun, H.J.; Wu, Z.Y.; Cao, L.; Zhu, M.Y.; Liu, T.T.; Guo, L.; Lin, Y.; Nie, X.W.; Bian, J.S. Hydrogen Sulfide: Recent Progression and Perspectives for the Treatment of Diabetic Nephropathy. Molecules 2019, 24, 2857. [CrossRef] 34. Magierowski, M.; Magierowska, K.; Surmiak, M.; Hubalewska-Mazgaj, M.; Kwiecien, S.; Wallace, J.L.; Brzozowski, T. The effect of hydrogen sulfide-releasing naproxen (ATB-346) versus naproxen on formation of stress-induced gastric lesions, the regulation of systemic inflammation, hypoxia and alterations in gastric microcirculation. J. Physiol. Pharmacol. 2017, 68, 749–756. [PubMed] 35. Cacanyiova, S.; Berenyiova, A.; Balis, P.; Kristek, F.; Grman, M.; Ondrias, K.; Breza, J.; Breza, J., Jr. Nitroso-sulfide coupled signaling triggers specific vasoactive effects in the intrarenal arteries of patients with arterial hypertension. J. Physiol. Pharmacol. 2017, 68, 527–538. [PubMed] 36. Cacanyiova, S.; Berenyiova, A.; Kristek, F.; Drobna, M.; Ondrias, K.; Grman, M. The adaptive role of nitric oxide and hydrogen sulphide in vasoactive responses of thoracic aorta is triggered already in young spontaneously hypertensive rats. J. Physiol. Pharmacol. 2016, 67, 501–512. [PubMed] 37. Berenyiova, A.; Drobna, M.; Cebova, M.; Kristek, F.; Cacanyiova, S. Changes in the vasoactive effects of nitric oxide, hydrogen sulfide and the structure of the rat thoracic aorta: The role of age and essential hypertension. J. Physiol. Pharmacol. 2018, 69, 10–26. [CrossRef] 38. Zhao, W.; Zhang, J.; Lu, Y.; Wang, R. The vasorelaxant effect of H2S as a novel endogenous gaseous K(ATP) channel opener. EMBO J. 2001, 20, 6008–6016. [CrossRef][PubMed] 39. Pechanova, O.; Bernatova, I.; Pelouch, V.; Simko, F. Protein remodelling of the heart in NO-deficient hypertension: The effect of captopril. J. Mol. Cell Cardiol. 1997, 29, 3365–3374. [CrossRef]