Effects of Duodenal 5-Hydroxytryptophan Perfusion on Melatonin Synthesis in GI Tract of Sheep

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Article Effects of Duodenal 5-Hydroxytryptophan Perfusion on Melatonin Synthesis in GI Tract of Sheep

Jun Pan 1,†, Fengming Li 1,†, Caidie Wang 1, Xiaobin Li 1, Shiqi Zhang 1, Wenjie Zhang 1, Guodong Zhao 1, Chen Ma 1, Guoshi Liu 2 and Kailun Yang 1,*,†

1 College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China; [email protected] (J.P.); [email protected] (F.L.); [email protected] (C.W.); [email protected] (X.L.); [email protected] (S.Z.); [email protected] (W.Z.); [email protected] (G.Z.); [email protected] (C.M.) 2 College of Animal Science and Technology, China Agricultural University, Beijing 100083, China; [email protected] * Correspondence: [email protected]; Tel.: +86-099-1876-2603 † The authors contributed equally to this work.

Abstract: The purpose of this study is to investigate the potential effects of 5-hydroxytryptophan (5-HTP) duodenal perfusion on melatonin (MT) synthesis in the gastrointestinal (GI) tract of sheep. 5-hydroxytryptophan is a precursor in the melatonin synthetic pathway. The results showed that this method signiﬁcantly increased melatonin production in the mucosa of all segments in GI tract including duodenum, jejunum, ileum, cecum and colon. The highest melatonin level was identiﬁed in the colon and this indicates that the microbiota located in the colon may also participate in the

melatonin production. In addition, portion of the melatonin generated by the GI tract can pass the liver metabolism and enters the circulation via portal vein. The current study provides further

Citation: Pan, J.; Li, F.; Wang, C.; Li, evidence to support that GI tract is the major site for melatonin synthesis and the GI melatonin also X.; Zhang, S.; Zhang, W.; Zhao, G.; contributes to the circulatory melatonin level since plasma melatonin concentrations in 5-HTP treated Ma, C.; Liu, G.; Yang, K. Effects of groups were signiﬁcantly higher than those in the control group. In conclusion, the results show Duodenal 5-Hydroxytryptophan that 10–50 mg of 5-HTP ﬂowing into the duodenum within 6 h effectively improve the production of Perfusion on Melatonin Synthesis in melatonin in the GI tract and melatonin concentration in sheep blood circulation during the day. GI Tract of Sheep. Molecules 2021, 26, 5275. https://doi.org/10.3390/ Keywords: sheep; intestinal mucosa; 5-hydroxytryptophan; melatonin; gastrointestinal track molecules26175275

Academic Editor: Raffaele Capasso 1. Introduction Received: 4 August 2021 Melatonin (MT) has multiple biological functions in mammals, including sleep pro- Accepted: 28 August 2021 Published: 31 August 2021 motion, immunoregulation, and antibacterial, anti-inﬂammatory, as well as antioxidative activities [1–4]. Since the ﬁrst isolation of MT from bovine pineal gland [5], MT was believed

Publisher’s Note: MDPI stays neutral to be exclusively produced in the pineal gland until MT was detected in the retina, Hard- with regard to jurisdictional claims in erian gland, cerebellum [6] and in the intestinal chromaffin cells [7]. These observations published maps and institutional affil- have expanded the melatonin synthetic sites to extrapineal tissues and organs. Currently, it iations. has been reported that melatonin is synthesized by mitochondria and many functions of melatonin is also mediated by the mitochondria [8]. As a result, virtually, every cell with mitochondria has the capacity to synthesize melatonin. For example, the total amounts of MT produced in the gastrointestinal tract (GI Tract) were estimated to be approximately 400 times more than that produced in the pineal gland [9]. As to its synthesis, tryptophan Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. (Trp) is its original precursor of melatonin in mammals [10]. First, Trp is decarboxylated to This article is an open access article 5-hydroxytryptophan (5-HTP) by the tryptophan hydroxylase (TPH) [11], and then 5-HTP distributed under the terms and is transformed into serotonin (5-HT) by aryl amino acid decarboxylase (AADC), and 5-HT is conditions of the Creative Commons converted to N-acetyl-5-hydroxytryptamine by a rate-limiting enzymes, arylalkylamine-N- Attribution (CC BY) license (https:// acetyltransferase (SNAT, previously AANAT), finally, the N-acetyl-5-hydroxytryptamine is creativecommons.org/licenses/by/ converted to melatonin by N-acetyl-5-hydroxytryptamine methyltransferase (ASMT, previ- 4.0/). ously HIOMT) [12]. Melatonin synthetic pathway in mammals is illustrated in the Figure1 .

Molecules 2021, 26, 5275. https://doi.org/10.3390/molecules26175275 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 14

decarboxylase (AADC), and 5-HT is converted to N-acetyl-5-hydroxytryptamine by a rate-limiting enzymes, arylalkylamine-N-acetyltransferase (SNAT, previously AANAT), finally, the N-acetyl-5-hydroxytryptamine is converted to melatonin by Molecules 2021, 26, 5275 N-acetyl-5-hydroxytryptamine methyltransferase (ASMT, previously HIOMT) 2[12] of 13. Melatonin synthetic pathway in mammals is illustrated in the Figure 1.

FigureFigure 1. 1. MelatoninMelatonin synthetic synthetic pathway pathway in inmammals. mammals. TPH: TPH: tryptophan tryptophan hydroxylase hydroxylase,, AADC: AADC: aryl aryl aminoamino acid acid decarboxylase decarboxylase,, SNAT: SNAT: serotonin serotonin N N-acetyltrasferase,-acetyltrasferase, ASMT: ASMT: N N-acetyltraptamine-acetyltraptamine me- methyl- thyltrasferase,trasferase, MAO: MAO: monoamine monoamine oxidase. oxidase.

MTMT plays plays important important roles roles in in the the improvement improvement of of the the yields yields and and quality quality of of animal animal products.products. For For example, example, it it improves improves the the meat meat quality quality and andquantity quantity of cashmere of cashmeregoats [ goats13,14] , [13,14]reduces, reduces somatic somatic cell count cell count and mastitis and mastitis in dairy in dairy cattle cattle to improve to improve the the quality quality of theof themilk milk [15 ,[15,16]16]. Currently,. Currently, in husbandryin husbandry industrial, industrial, the the methods methods used used to regulationto regulation of MTof MTlevels levels in animals in animals are primarily are primarily to alter to the alter light/dark the light/dark cycle ofcycle the of environments the environments [17,18], [17,18]or by, implantationor by implantation [19,20], [19,20] injection, injection [21], and [21] oral, and administration oral administration [22] of [22] MT. of As MT. to As the exogenous melatonin administration, several factors should be considered including its to the exogenous melatonin administration, several factors should be considered in- bioavailability, acting duration, economic benefit, and delivery methods [22]. Addition- cluding its bioavailability, acting duration, economic benefit, and delivery methods [22]. ally, exogenous MT supplementation seems to reduce endogenous MT synthesis and to Additionally, exogenous MT supplementation seems to reduce endogenous MT synthe- down-regulate expression of the MT receptors [23–25]. Thus, researchers have tried to sis and to down-regulate expression of the MT receptors [23–25]. Thus, researchers have improve melatonin production by manipulating the availability of MT precursor, that is, to tried to improve melatonin production by manipulating the availability of MT precursor, give Trp to animals. The results are variable. Some studies indicated that only 1–2% of Trp that is, to give Trp to animals. The results are variable. Some studies indicated that only ingested were converted into 5-HT depending on the species [26]. Several studies have 1–2% of Trp ingested were converted into 5-HT depending on the species [26]. Several shown that Trp supply increases blood MT levels in the tested animals [27–30]. However, studies have shown that Trp supply increases blood MT levels in the tested animals this increase only occurs nocturnally, or at certain time points [31,32], while others reported [27–30]. However, this increase only occurs nocturnally, or at certain time points [31,32], the negative results with no increased melatonin production [33,34]. Specifically to sheep, while others reported the negative results with no increased melatonin production a study showed that 5-HTP supply increased the MT synthesis, but Trp supply failed to [33,34]achieve. Specifically this goal [35 to]. sheep, Due to a thestudy uncertainty showed that of Trp 5-HTP supply supply on melatonin increased productionthe MT syn- in thesis,sheep, but we Trp select supply the 5-HTP failed as to the achieve precursor this goal to feed [35] sheep. Due and to the attempt uncertainty to identify of Trp whether sup- plythis on treatment melatonin can production improve the in melatoninsheep, we productionselect the 5- inHTP sheep. as the Indeed, precursor we have to feed found sheep that andfeeding attempt 5-HTP to identify increases whether MT content this oftreatment blood plasma can improve from the the jugular melatonin vein inproduction sheep [36, 37in ] sheep.which Indeed, mainly reflectswe have the found pineal tha originatedt feeding melatonin5-HTP increases [9,38]. FewMT content studies haveof blood investigated plasma fromthe effects the jugular of 5-HTP vein supply in sheep on the[36,37] GI tract which melatonin mainly synthesisreflects the [39 pineal], especially, originated the different mela- toninsegments [9,38] of. Few GI tract.studies GI have tract investigated is considered the as effects one of of the 5 largest-HTP supply extrapineal on the melatonin GI tract melatoningenerating synthesis site and melatonin[39], especially, plays the different important segments physiological of GIfunctions tract. GI tract on it is [10 consid-]. Thus, eredin the as current one of study,the largest we focus extrapineal whether melatonin 5-HTP supplementation generating site also and increases melatonin the plays melatonin the importantsynthesis inphysiological the different functions sections ofon GI it tract.[10]. T Ashus, we in know the current that the study, melatonin we focus in portal whether vein 5blood-HTP supplementation plasma is the indicator also increases of GI generated the melaton melatoninin synthesis and the in melatonin the different injugular sections vein of mainly represents the pineal generated melatonin [9,38]. For comparative purpose, both the portal vein and the jugular vein blood were collected. The results from the current study will answer the following questions: (1) Whether duodenal 5-HTP supplementation will increase the GI melatonin synthesis? (2) If it is so, does the GI synthesized melatonin contribute to the circulatory melatonin? Molecules 2021, 26, x FOR PEER REVIEW 3 of 14

GI tract. As we know that the melatonin in portal vein blood plasma is the indicator of GI generated melatonin and the melatonin in jugular vein mainly represents the pineal generated melatonin [9,38]. For comparative purpose, both the portal vein and the jugular vein blood were collected. The results from the current study will answer the following questions: (1) Whether duodenal 5-HTP supplementation will increase the GI melatonin synthesis? (2) If it is so, does the GI synthesized melatonin contribute to the circu- Molecules 2021, 26, 5275 latory melatonin? 3 of 13

2. Results 2.2.1. Results Effects of Different Doses of 5-HTP Duodenal Perfusion on 5-HTP Content in Sheep 2.1.Intestinal Effects Mucosa of Different Doses of 5-HTP Duodenal Perfusion on 5-HTP Content in Sheep IntestinalThe Mucosa levels of 5-HTP in general intestinal tract mucosa were significantly increased in bothThe 10 and levels 50 of mg 5-HTP duodenal in general 5-HTP intestinal perfused tractgroups mucosa compared were to signiﬁcantly the control increasedgroup (p < in0.01) both (Figure 10 and 2 50A). mg 5-HTP duodenal content 5-HTPs in the perfused jejunum, groups ileum, compared cecum, and to colon the control mucosa group were (pincreased< 0.01) (Figure by 35.56%2A). 5-HTP(p < 0.01), contents 30.19% in ( thep < jejunum,0.01), 2.85% ileum, (p > cecum,0.05), and and 22.40% colon mucosa(p < 0.05), wererespectively, increased in by the 35.56% 10 mg (p 5<-HTP 0.01), perfused 30.19% (p group< 0.01), compared 2.85% (p > to 0.05), controls and 22.40%(Figure( 2pB<). 0.05)In the, respectively,50 mg group, in the 105-H mgTP 5-HTPlevels in perfused duodenum, group jejunum, compared ileum, to controls cecum, (Figure and colon2B). Inmucosa the 50increa mg group,sed by the44.17 5-HTP (p < levels0.01), 28.00% in duodenum, (p < 0.05), jejunum, 30.26% ileum, (p < 0.01), cecum, 30.53% and colon(p < 0.01), mucosa and increased28.14% (p by < 0.0 44.175), (respectively.p < 0.01), 28.00% The highest (p < 0.05), concentration 30.26% (p < of 0.01), 5-HTP 30.53% in 50 ( pmg< 0.01),group and was 28.14%observed (p < in 0.05), the duodenum respectively. mucosa The highest (Figure concentration 2B). of 5-HTP in 50 mg group was observed in the duodenum mucosa (Figure2B).

Figure 2. Effects of different doses of 5-HTP duodenal perfusion on 5-HTP content in sheep intestinal mucosa. (A) 5-HTP levelsFigure in 2. general Effects intestinalof different tract doses mucosa. of 5-HTP (B) duodenal 5-HTP levels perfusion in the on different 5-HTP content intestinal in segmentsheep intestinal mucosa. mucosa. The data (A) were5-HTP expressedlevels in general as LSM intestinal± SEM ( ntract= 6). mucosa. Small ( lettersB) 5-HTP indicate levelsp in< the 0.05, different capital lettersintestinal indicate segmentp < mucosa 0.01 vs.. The their data respective were ex- controlpressed groups. as LSM ± SEM (n = 6). Small letters indicate p < 0.05, capital letters indicate p < 0.01 vs. their respective control groups. 2.2. Effects of Different Doses of 5-HTP Duodenal Perfusion on the Blood 5-HTP Levels Collected from2.2. DifferentEffects of BloodDifferent Vessels Doses of 5-HTP Duodenal Perfusion on the Blood 5-HTP Levels Collected fromThe Different 5-HTP Blood concentrations Vessels in circulation blood plasma were signiﬁcantly increased Molecules 2021, 26, x FOR PEER REVIEWwith 10The and 5- 50HTP mg concentration 5-HTP duodenals in infusioncirculation (p < blood 0.01) (Figureplasma3 A).were It seemedsignificantly4 thatof 14 the increased rapid increasedwith 10 and 5-HTP 50 concentrationsmg 5-HTP duodenal in circulation infusion blood (p < also0.01) quickly (Figure reached 3A). It its seemed steady that status the withinrapid oneincreased hourafter 5-HTP perfusion concentrations (Figure 3inB). circulation blood also quickly reached its steady status within one hour after perfusion (Figure 3B).

Figure 3. Effects of different doses of 5-HTP duodenal perfusion on circulating blood 5-HTP content. Figure 3. Effects of different doses of 5-HTP duodenal perfusion on circulating blood 5-HTP content.(A )(A 5-HTP) 5-HTP levels levels in the the circulating circulating blood blood plasma. plasma. (B) Dynamic (B) Dynamic changes changesof 5-HTP levels of 5-HTP in the levels in the circulatingcirculating blood blood plasma plasma with time. with The time. data The were data expres weresed expressed as LSM ± SEM as LSM (n = ±6).SEM Small ( nletters= 6). Small letters indicateindicate p < p0.05,< 0.05, capital capital letters letters indicate indicate p < 0.01p vs.< 0.01their vs. respective their respective control groups. control groups.

In addition to the circulation blood, the 5-HTP concentrations in blood of portal vein, carotid artery, jugular vein, and posterior vena cava were also significantly increased compared to their respective controls (p < 0.01) (Figure 4A–D). While 5-HTP concentrations among the perfused groups did not exhibit significant differences during the process of 5-HTP duodenal perfusion (p > 0.05) (Figure 4A–D).

Figure 4. Effects of different doses of 5-HTP duodenal perfusion on blood 5-HTP levels of different blood vessels. (A) portal vein, (B) carotid artery, (C) jugular vein and (D) posterior vena cava. The data were expressed as LSM ± SEM (n = 6). The capital letters indicate p < 0.01 vs. control.

As to the timely dynamic changes of plasma 5-HTP concentration from portal vein, the 5-HTP level rapidly increased to 257.5 ng/mL after 1 h of perfusion, and then stabilized at the range from 212.9 to 242.5 ng/mL during the entire experimental period and its level at 4 h was significantly higher than that of the control (p < 0.05). However, 2 h after completion of perfusion, the concentrations of 5-HTP were declined in both of two

Molecules 2021, 26, x FOR PEER REVIEW 4 of 14

Figure 3. Effects of different doses of 5-HTP duodenal perfusion on circulating blood 5-HTP content. (A) 5-HTP levels in the circulating blood plasma. (B) Dynamic changes of 5-HTP levels in the Molecules 2021, 26, 5275 4 of 13 circulating blood plasma with time. The data were expressed as LSM ± SEM (n = 6). Small letters indicate p < 0.05, capital letters indicate p < 0.01 vs. their respective control groups.

InIn addition addition to to the circulation circulation blood, blood, the the 5-HTP 5-HTP concentrations concentration ins bloodin blood of portal of portal vein, vein,carotid carotid artery, artery, jugular jugular vein, vein, and posterior and posterior vena vena cava werecava were also signiﬁcantly also significantly increased in- creasedcompared compared to their respective to their respective controls ( p controls< 0.01) (Figure (p < 0.01)4A–D). (Figure While 4 5-HTPA–D). concentrationsWhile 5-HTP concentrationamong the perfuseds among groupsthe perfused did not groups exhibit did signiﬁcant not exhibit differences significant during differences the process during of the5-HTP process duodenal of 5-HTP perfusion duodenal (p >perfusion 0.05) (Figure (p > 40.05)A–D). (Figure 4A–D).

Figure 4. Effects of different doses of 5-HTP duodenal perfusion on blood 5-HTP levels of different Figure 4. Effects of different doses of 5-HTP duodenal perfusion on blood 5-HTP levels of different bloodblood vessels. vessels. (A (A) portal) portal vein, vein, (B ()B carotid) carotid artery, artery, (C (C) jugular) jugular vein vein and and (D (D) posterior) posterior vena vena cava. cava. The The datadata were were expressed as as LSM LSM± ±SEM SEM (n (=n 6).= 6). The The capital capital letters letters indicate indicatep < 0.01 p < vs. 0.01 control. vs. control. As to the timely dynamic changes of plasma 5-HTP concentration from portal vein, As to the timely dynamic changes of plasma 5-HTP concentration from portal vein, the 5-HTP level rapidly increased to 257.5 ng/mL after 1 h of perfusion, and then stabilized the 5-HTP level rapidly increased to 257.5 ng/mL after 1 h of perfusion, and then stabi- at the range from 212.9 to 242.5 ng/mL during the entire experimental period and its lized at the range from 212.9 to 242.5 ng/mL during the entire experimental period and its level at 4 h was signiﬁcantly higher than that of the control (p < 0.05). However, 2 h after level at 4 h was significantly higher than that of the control (p < 0.05). However, 2 h after completion of perfusion, the concentrations of 5-HTP were declined in both of two 5-HTP completion of perfusion, the concentrations of 5-HTP were declined in both of two perfusion groups. In addition, no signiﬁcant differences were observed in the two 5-HTP perfusion groups. The very similar pattern of the plasma 5-HTP was observed in the blood from carotid artery, jugular vein, and posterior vena cava, respectively (Figure5B–D).

2.3. Effects of Different Doses of 5-HTP Perfusion on MT Content in Sheep Intestinal Mucosa The contents of MT in entire intestinal tract mucosa were significantly increased by 23.16% (p < 0.05) and 49.70% (p < 0.01) in 10 and 50 mg duodenal 5-HTP perfused groups, respectively compared to the control (Figure6A). MT contents in different GI tract segments also significantly increased including jejunum (p < 0.05), ileum (p < 0.01), cecum (p < 0.01), and colon (p < 0.01) mucosa in 50 mg 5-HTP duodenal perfused group compared to the control group (Figure6B), while MT content levels in the ileum (p < 0.01) and cecum (p < 0.05) mucosa in 10 mg 5-HTP perfused groups were also significantly increased compared to their control group. The highest content of MT was found in the colon mucosa of 50 mg 5-HTP perfused group (around 7.8 pg/mg tissue). Molecules 2021, 26, x FOR PEER REVIEW 5 of 14

5-HTP perfusion groups. In addition, no significant differences were observed in the two 5-HTP perfusion groups. The very similar pattern of the plasma 5-HTP was observed in the blood from carotid artery, jugular vein, and posterior vena cava, respectively (Figure 5B–D).

Molecules 2021, 26, x FOR PEER REVIEW 5 of 14

5-HTP perfusion groups. In addition, no significant differences were observed in the two

Molecules 2021, 26, 5275 5-HTP perfusion groups. The very similar pattern of the plasma 5-HTP was observed5 of in 13 the blood from carotid artery, jugular vein, and posterior vena cava, respectively (Figure 5B–D).

Figure 5. Effects of different levels of duodenal 5-HTP perfusion on the timely dynamic changes of blood 5-HTP in different blood vessels: (A) portal vein, (B) carotid artery, (C) jugular vein and (D) posterior vena cava. The data were expressed as LSM ± SEM (n = 6). Small letters indicate p < 0.05 vs. their respective controls.

toFigure the control 5. Effects group of different (Figure levels6B), while of duodenal MT content 5-HTP levels perfusion in the on theileum timely (p < dynamic 0.01) and changes ce- Figure 5. Effects of different levels of duodenal 5-HTP perfusion on the timely dynamic changes of cumof blood (p < 0.0 5-HTP5) mucosa in different in 10 blood mg 5 vessels:-HTP perfused (A) portal groups vein, ( Bwere) carotid also artery, signifi (Ccantly) jugular increased vein and blood 5-HTP in different blood vessels: (A) portal vein, (B) carotid artery, (C) jugular vein and (D) compared(D) posterior to their vena control cava. The group. data The were highest expressed content as LSM of ±MTSEM was (n found= 6). Small in the letters colon indicate mu- cosaposterior of 50 vena mg 5cava.-HTP The perfused data were group expressed (around as L SM7.8 ±pg/mg SEM ( ntissue). = 6). Small letters indicate p < 0.05 vs.p

2.3. Effects of Different Doses of 5-HTP Perfusion on MT Content in Sheep Intestinal Mucosa The contents of MT in entire intestinal tract mucosa were significantly increased by 23.16% (p < 0.05) and 49.70% (p < 0.01) in 10 and 50 mg duodenal 5-HTP perfused groups, respectively compared to the control (Figure 6A). MT contents in different GI tract segments also significantly increased including jejunum (p < 0.05), ileum (p < 0.01), cecum (p < 0.01), and colon (p < 0.01) mucosa in 50 mg 5-HTP duodenal perfused group compared to the control group (Figure 6B), while MT content levels in the ileum (p < 0.01) and cecum (p < 0.05) mucosa in 10 mg 5-HTP perfused groups were also significantly increased compared to their control group. The highest content of MT was found in the colon mucosa of 50 mg 5-HTP perfused group (around 7.8 pg/mg tissue).

Figure 6. Effects of different doses of 5-HTP duodenal perfusion on MT content in sheep intestinal mucosa. (A). MT level in entire intestinal tract mucosa, (B). MT levels in mucosa of duodenum, jejunum, ileum cecum and colon, respectively. The data were expressed as LSM ± SEM (n = 6). Small letters indicate p < 0.05, capital letters indicate p < 0.01 vs. their respective control groups.

2.4. Effects of Different Doses of 5-HTP Duodenal Perfusion on Blood MT Levels from Different Blood Vessels The circulating blood plasma MT concentrations were signiﬁcantly increased in 10 and 50 mg 5-HTP duodenal perfused groups, respectively, compared to the control group (p < 0.01), and MT concentration in 50 mg group was also higher than that in 10 mg group (p < 0.05). The highest melatonin concentration in the circulating blood was achieved at 4 h after perfusion and the much higher melatonin concentrations were preserved until 2 h after the ending of perfusion (Figure7B).

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Figure 6. Effects of different doses of 5-HTP duodenal perfusion on MT content in sheep intestinal mucosa. (A). MT level in entire intestinal tract mucosa, (B). MT levels in mucosa of duodenum, jejunum, ileum cecum and colon, respectively. The data were expressed as LSM ± SEM (n = 6). Small letters indicate p < 0.05, capital letters indicate p < 0.01 vs. their respective control groups.

2.4. Effects of Different Doses of 5-HTP Duodenal Perfusion on Blood MT Levels from Different Blood Vessels The circulating blood plasma MT concentrations were significantly increased in 10 and 50 mg 5-HTP duodenal perfused groups, respectively, compared to the control group (p < 0.01), and MT concentration in 50 mg group was also higher than that in 10 mg group (p < 0.05). The highest melatonin concentration in the circulating blood was Molecules 2021, 26, 5275 achieved at 4 h after perfusion and the much higher melatonin concentrations were pre-6 of 13 served until 2 h after the ending of perfusion (Figure 7B).

Figure 7. Effects of different doses of 5-HTP duodenal perfusion on circulating blood MT concentrations. Figure 7. Effects of different doses of 5-HTP duodenal perfusion on circulating blood MT concentrations(A) Circulating. (A) Circulating blood melatonin blood melatonin concentration. concentration. (B) The timely (B) The dynamic timely changes dynamic of circulatingchanges of blood circu- MT latingconcentration blood MT after concentration 5-HTP duodenal after 5 perfusion.-HTP duodenal The data perfusion. were expressed The data as were LSM expressed± SEM (n as= 6).LSM Small ± SEMletters (n = indicate 6). Smallp < letters 0.05, capitalindicate letters p < 0.05, indicate capitalp < letters 0.01 vs. indicate their respective p < 0.01 vs. control their groups.respective control groups. The blood plasma MT concentrations in the portal vein, carotid artery, jugular vein andThe posterior blood venaplasma cava, MT were concentrations also signiﬁcantly in the higherportal invein, the carotid 10 and 50artery, mg treated jugular groups vein Molecules 2021, 26, x FOR PEER REVIEW 7 of 14 andthan posterior those in vena their respective cava, were control also significantly group (p < 0.01) higher (Figure in the8A–D). 10 and In addition, 50 mg treated plasma groupsMT concentrations than those in intheir the respective 50 mg group control were group also higher (p < 0.01) than (Figure thoseof 8A the–D 10). In mg addition, group in plasmathe portal MT vein,concentrations carotid artery, in the jugular 50 mg vein group and were posterior also venahigher cava, than respectively those of the (p 10> 0.05).mg group in the portal vein, carotid artery, jugular vein and posterior vena cava, respectively (p > 0.05).

FigureFigure 8. 8. BloodBlood MT MT concentrations concentrations in different in different blood blood vessels vessels after after5-HTP 5-HTP duodenal duodenal perfusion. perfusion. (A) portal(A) portal vein, vein,(B) carotid (B) carotid artery, artery, (C) jugular (C) jugular vein and vein (D and) posterior (D) posterior vena cava. vena The cava. data The were data ex- were pressedexpressed as LSM as LSM ± SEM± SEM (n = ( n6).= The 6). Thecapital capital letters letters indicate indicate p < 0.01p < 0.01vs. control. vs. control.

TheThe timely dynamic dynamic changes changes of of blood blood melatonin melatonin concentrations concentrations in the in different the different blood bloodvessels vessels were also were monitored. also monitored. For example, For example, in the portal in the vein, portal the highestvein, the blood highest melatonin blood melatoninconcentration concentration (63.5 pg/mL) (63.5 was pg/mL) achieved was at achieved the 4 h after at the 5-HTP 4 h after perfusion 5-HTP which perfusion were whichhigher were than higher that of than control that group of control (p < 0.05), group and (p these< 0.05) levels, and ﬂuctuatedthese levels in fluctuated the range ofin 48.9the rangeto 63.5 of pg/mL 48.9 to during 63.5 pg/mL the entire during perfusion the entire period perfusion until to theperiod 2 h afteruntil perfusion to the 2 h terminated, after per- fusionwhile theseterminated levels, ofwhile control these were levels 40.9 of tocontrol 46.9 pg/mL were 40.9 (Figure to 46.99A). pg/mL The blood (Figure melatonin 9A). The bloodconcentrations melatonin in concentrati the carotidons artery, in the jugular carotid vein artery, and jugular posterior vein vena and cava posterior showed vena the cavasimilar showed pattern the as similar in the portalpattern vein as in (Figure the portal9B–D). vein (Figure 9B–D).

Molecules 2021, 26, x FOR PEER REVIEW 7 of 14

Figure 8. Blood MT concentrations in different blood vessels after 5-HTP duodenal perfusion. (A) portal vein, (B) carotid artery, (C) jugular vein and (D) posterior vena cava. The data were expressed as LSM ± SEM (n = 6). The capital letters indicate p < 0.01 vs. control.

The timely dynamic changes of blood melatonin concentrations in the different blood vessels were also monitored. For example, in the portal vein, the highest blood melatonin concentration (63.5 pg/mL) was achieved at the 4 h after 5-HTP perfusion which were higher than that of control group (p < 0.05), and these levels fluctuated in the range of 48.9 to 63.5 pg/mL during the entire perfusion period until to the 2 h after perfusion terminated, while these levels of control were 40.9 to 46.9 pg/mL (Figure 9A). The Molecules 2021, 26, 5275 blood melatonin concentrations in the carotid artery, jugular vein and posterior vena7 of 13 cava showed the similar pattern as in the portal vein (Figure 9B–D).

Figure 9. The timely dynamic changes of melatonin concentration in different blood vessels after 5-HTP duodenal perfusion. (A) Portal vein, (B) carotid artery, (C) jugular vein and (D) posterior vena cava. The data were expressed as LSM ± SEM (n = 6). Small letters indicate p < 0.05 vs. their respective control groups.

3. Discussion Whether the Trp supplementation can increase melatonin production in sheep is debatable [33]. Thus, in the current study, we avoided using Trp but selected another precursor of melatonin synthesis, 5-HTP, to test its effect on melatonin productions in sheep (Kazakh sheep). As we know that sheep are ruminants, if 5-HTP is orally given to sheep, it needs to pass the rumen in which digestive enzymes and microbiota may consume some of the 5-HTP. This will impact the bioavailability of 5-HTP. In this consideration, we have selected the duodenal perfusion to deliver the 5-HTP. The results showed that duodenal perfusion of 5-HTP signiﬁcantly increased the blood and GI tissue 5-HTP levels. This observation is consistent with previous reports. Studies have shown when sheep were orally given 111–222 mg/kg LBW (equivalent 5-HTP 50–100 mg/kg LBW) rumen-protected 5-HTP, it increased their jejunum, ileum, cecum, and colon mucosa and plasma 5-HTP concentrations; however, the dose they used was much higher than those used in this study [36,39,40]. It has been reported that high dose of 5-HTP intravenous administration (over 300 mg/d) leads to animal developing poisoning symptoms similar to 5-HT-induced toxicity [41]. In the pilot study, we have also found that if the 5-HTP duodenal perfusion dose in excessive of 50 mg/d (equivalent 1 mg/kg LBW) the sheep drastically reduced food intake or even refused to eat, and then, the sheep developed irritable and restless symptoms. While if the dose of 5-HTP was under 50 mg/d, no adverse effects were observed. Based on the results mentioned above, in the current study, 5-HTP doses selected were 10 and 50 mg/d which is 50–100 times lower than that used by Zhao et al. [36,39]. This difference may be due to the bioavailability of 5-HTP by the different delivery methods. 5-HTP orally administration has to pass the rumen and majority of it may be metabolized by the digestive enzymes or microbiota inhabited in the rumen. 5-HTP is a direct precursor of 5-HT and it can be transformed into 5-HT by aryl amino acid decarboxylase, without biochemical feedback or rate-limiting steps. In GI tract, this process mainly occurs in the intestinal chromafﬁn cells of the intestinal mucosa [40,42,43]. The increased concentration of 5-HT in the small intestine after 5-HTP treatment has also been reported in different species including rats [42] and humans with Parkinson’s Molecules 2021, 26, 5275 8 of 13

disease [44] or health subjects [45]. Our focus is whether this increased 5-HTP levels in local GI tissue and blood will be converted to melatonin since there is few investigations to deal this important question. Our results, for the first time, showed that the increased 5-HTP not only elevated the melatonin content in the GI tissue mucosa but also increase the circulatory melatonin levels such as in the blood from carotid artery. This is a long- lasting argument whether the GI track synthesized melatonin is attributed to the circulatory melatonin. It has been hypothesized that the locally synthesized melatonin would not release into the circulation but be consumed by the local tissues as the antioxidant [46]. Our results clearly showed that the GI track locally synthesized melatonin contribute to the circulatory melatonin. By collecting blood from different blood vessels, we try to identify the sources of melatonin in the blood. We observed in all animals, their melatonin levels in jugular vein are higher than that in the carotid artery. This indicated that brain tissue releases melatonin into blood. Several studies have also investigated the potential relationship between the melatonin levels of jugular vein and carotid artery. The results are not consistent. Bubenik et al. reported that 1–2 h after feeding, jugular vein melatonin only slightly increased with no significant difference with the melatonin in the carotid artery [38]. This is similar to the observation of Huether et al. with Trp orally administration [9]. In contrast, nocturnal concentration of MT was reduced after intramuscular injection of 5-HTP in quails [47]. This discrepancy may be species specific. In sheep, with intraperitoneal injection of 5-HTP the diurnal MT level in jugular vein was increased but no effect was observed on nocturnal MT level [35,48]. As to the melatonin levels in the portal vein, the results are consistent based on the previous observations. In pigs, the highest melatonin level was observed in the portal vein among carotid artery and posterior vena cava after feeding [38]. In chicken and rats, after oral administration of 300 mg/kg Trp, the portal vein melatonin was significantly higher than that in jugular vein [9]. Similarly, the concentration of MT in human portal vein was also higher than that in peripheral veins [49]. Our results are in consistent with the above-mentioned results. It is well documented that major portion of melatonin is metabolized in the liver by the CYP 1A2 to form the 6-hydroxymelatonin [50]. For example, studies reported that 92–97% of MT in male Sprague–Dawley rats was cleared up by the liver via a single passage [51]. This strongly suggest that much more melatonin has been produced in the GI tract before it reaches to the portal vein. Originally, intestinal chromaffin cells are believed to be the main site of intestinal MT synthesis [7]. The AA-NAT and HIOMT identified in rat intestinal chromaffin further support this observation [52]. However, following the discovery of mitochondria being the main site of melatonin synthesis [53], virtually all the cells in the GI tract have the capacity to synthesize melatonin. Our observations that mucosa of all the segments in GI tract including duodenum, jejunum, ileum, cecum and colon have increased melatonin content after 5-HTP perfusion support this idea. Very interestingly, the highest melatonin level was identified in the colon. This is not surprising due to the discovery that the microorganisms including the bacteria have the ability to synthesize melatonin [54], since the microbiota located in the colon also participate in the melatonin synthesis. The melatonin produced in GI tract is primarily functional locally to protect the gut from the oxidative stress and inflammation [55,56], but also can release into circulation for other systemic functions. The limitation of this study is that we have not explored whether the GI generated melatonin contribute to the normal melatonin circadian rhythm. This requires collecting blood samples during a 24 h interval. In the current study, the blood samples were only collected during an 8 h interval. However, to test the contribution of GI generated melatonin to melatonin circadian rhythm will be our future project. In conclusion, the results show that 5-HTP duodenal perfusion avoids the metabolism of this substance by the digestive enzymes and microbiota in the rumen and increases the bioavailability of 5-HTP. Therefore, duodenal supplementation with 10–50 mg 5-HTP within 6 h can effectively improve the production of melatonin in sheep, particularly in the GI tract of the sheep. Molecules 2021, 26, 5275 9 of 13

4. Material and Methods 4.1. Materials 5-HTP (purity, >98%) was purchased from Xi’an lvruquan Biotechnology Co., Ltd. (Xi’an, China).

4.1.1. Animals Local Kazakh sheep (Ovis aries) from Xinjiang, China, were selected and all animals were housed in the animal farmhouse. The experiments were conducted in Changji Huikang Animal Husbandry Co., Ltd., Xinjiang, China, from April to June in 2020. Dur- ing the experiment period, the local sunrise times were 06:23–06:41, sunset times were 21:33–21:56, the external environment temperature was 22–33 ◦C, and the temperature in the animal house was 25–30 ◦C. The animal study has been approved by the Animal Wel- fare and Ethics Committee of Xinjiang Agricultural University, and the approved protocol number was 2020022.

4.1.2. Experimental Design Kazakh sheep (n = 18), aged 15–18 months and weighing (49.80 ± 1.96 kg) were used in the experiment. Six months after the installation of duodenal ﬁstula, the 18 sheep were divided into three groups with even numbers using a single factor randomized block design based on the sheep body weight, as follows: Control group: duodenal perfusion with 400 mL normal saline; 5-HTP treated group I and II: duodenal perfusion with 400 mL normal saline containing 10 mg and 50 mg 5-HTP, respectively.

4.1.3. Animal Feeding and Management The sheep were kept in a single/pen (i.e., 18 pens in total) with free access to drinking water. Sheep diet composition and nutrition levels (presented in Table1) were formulated according to National Research Council (NRC) version 2007 (NRC07). Each sheep was fed with concentrate formula (600 g/d), alfalfa hay (500 g/d), and wheat straw (300 g/d). The roughage was crushed to 2–3 cm and mixed in proportion. The diet was fed with roughage ﬁrst, followed by concentrate formula. For the ﬁrst 6 days, sheep were fed twice a day, and on the seventh day, they were deprived of diet and water from 6:00 a.m.

Table 1. Diet composition and nutrient levels (%).

Ingredient Ratio Items Nutrient Levels Maize 25.00 Dry matter DM 92.01 Wheat bran 6.00 Crude protein CP 16.39 Soybean meal 10.00 Ether extract EE 9.91 Cottonseed meal 6.50 Neutral detergent ﬁber NDF 52.89 Alfalfa hay 33.33 Acid detergent ﬁber ADF 36.22 Wheat straw 16.67 Ash 7.99 Premix 2.50 Calcium Ca 0.50 Total 100 Phosphorus P 0.35

4.2. Duodenal Perfusion and Sample Collection 4.2.1. Duodenal Perfusion 5-HTP (10 mg = 0.02 mg/kg LBW and 50 mg = 1 mg/kg LBW) were accurately weighed and dissolved in 400 mL normal saline. The solution was perfused via the duodenal fistula at a rate of 1.11 mL/min (22 drops/min) beginning at 7:30 a.m. after first feeding. The perfusion flow rate was checked and corrected every 30 min, to ensure the stable flow. The perfusion process lasted approximately 6 h/d and for consecutively 7 days.

4.2.2. Animal Surgery and Blood Collection At 6:00 a.m. of day 7, the animals were anesthetized with 0.02 mL/kg xylazine hydrochloride, a 15–20 cm abdominal incision was made at 1 cm from the posterior edge Molecules 2021, 26, 5275 10 of 13

of the thirteen ribs, and the hepatic portal vein, posterior vena cava, carotid artery, and jugular vein were isolated and catheterized for blood collection. The methods were brieﬂy described as following. After abdominal incision, the hepatic portal lymph node was located and a trocar inserted into the hepatic portal vein at a 45◦ angle anterior to the hepatic portal lymph node. Then, the internal needle was withdrawn and the cannula Molecules 2021, 26, x FOR PEER REVIEWinserted 1–2 cm forward, with the tip of the cannula 0.5 cm away from the liver. A similar11 of 14 method was used to insert the posterior vena cava cannula, with the tip located below the head of the kidney. An incision was made in the neck skin and the jugular vein and carotid artery intubated in the same way, with the tip of the cannula located 10 cm away from the fromhead. the The head. blood The was blood collected was atcollected 7:30 a.m. at before7:30 a.m. duodenal before perfusion.duodenal perfusion. Then, it started Then, the it startedregular the 6 h ofregular 5-HTP 6 duodenalh of 5-HTP perfusion. duodenal After perfusion. the completion After th ofe thecompletion 6 h of perfusion, of the 6 5h mL of perfusion,of blood were 5 mL collected of blood from were each collected of the from four bloodeach of vessels the four mentioned blood vessels above mentioned at the time abovepoints at of the 1, 2, time 3, 4, points 5, 6, 7, of and 1, 2, 8 h,3, respectively.4, 5, 6, 7, and The 8 h, blood respectively. samples The were blood rapidly samples transferred were rapidlyto heparin transferred sodium to anticoagulant heparin sodium tubes anticoagulant and centrifuged tubes atand 1350 centrifuged× g for 15 at min, 1350 plasma × g for 15samples min, plasma were collected samples into were cryopreservation collected into cryop tubesreservation and stored tubes at −20 and◦C storedfor future at −20 use °C to fordetect future the MTuse and to detect 5-HTP the concentrations. MT and 5-HTP Blood concentrations. and mucosa sampleBlood and collection mucosa procedure sample collectionare illustrated procedure in Figure are 10illustrated. in Figure 10.

Figure 10. Blood and mucosa sample collection procedure. Figure 10. Blood and mucosa sample collection procedure. 4.2.3. Mucosal Sample Collection 4.2.3. Mucosal Sample Collection Two hours after completion of 6 h of 5-HTP perfusion, the two ends of the junction of abomasumTwo hours and after rectum completion were ligatedof 6 h of with 5-HTP sutures perfusion, under the anesthesia. two ends Then, of the the junction sheep ofintestines abomasum were and quickly rectum removed, were ligated and the with duodenum, sutures under jejunum, anesthesia. ileum, cecum, Then, colon,the sheep and intestinesrectum were were excised, quickly respectively. removed, and The the contents duodenum, of each jejunum, intestinal ileum, segment cecum, were colon, gently and rectumtransferred were into excised, a clean respectively. beaker. Then, The each contents intestinal of each segment intestinal was cutsegment longitudinally were gently and transferredwashed five into times a clean with normalbeaker. saline,Then, each and theintestinal surface segment of the mucosa was cut was longitudinally dried by torching and washedto filter paper. five times The intestinal with normal mucosa saline, was and scraped the surface with a slide, of the and mucosa then transferred was dried into by torchian RNAse-freeng to filter cryopreservationpaper. The intestinal tube, mucosa and immediately was scraped storage with a atslide, liquid and nitrogen then trans- for ferredfuture into use. an RNAse-free cryopreservation tube, and immediately storage at liquid nitrogen for future use. 4.3. Measurement of 5-HTP and MT 4.3. MeasurementPlasma sample of 5- pretreatment:HTP and MT After thawing, plasma samples were fully mixed, cen- trifugedPlasma at 1000 sample× g pretreatment:at ambient temperature After thawing, for 10 plasma min, and samples then supernatantswere fully mixed, were centrifugedcollected for at measurement. 1000× g at ambient temperature for 10 min, and then supernatants were collectedIntestinal for measurement. mucosa sample pretreatment: After thawing, mucosa sample were cen- trifugedIntestinal at 1000 mucosa× g for sample 10 min, pretreatment: and cleaned Afterwith filter thawing, paper mucosa to remove sample the remainingwere cen- trifugedblood and at fluid.1000× g 1 mLfor 1 normal0 min, saline,and cleaned containing with filter 20 µL paper of 0.05 to mol/Lremove acetic the remaining acid were bloodadded and to the fluid. sample 1 mL and normal fully homogenized saline, containing with a 20 homogenizer µL of 0.05 and mol/L then, acetic sonicated acid were with × addedultrasound to the for sample 20 min. and The fully samples homogenized were centrifuged with a athomogenizer 1000 g at ambient and then, temperature sonicated withfor 10 ultrasound min, the supernatants for 20 min. T werehe samples collected. were The centrifuged pH of the at supernatant 1000× g at ambient was adjusted temper- to µ ature7.4 using for 10 0.05 min, mol the NaOH, supernatants and a 10 wereL aliquot collected. were The used pH for of protein the supernatant quantification. was One ad- gram protein of each sample was used for plasma and intestinal mucosa MT and 5-HTP justed to 7.4 using 0.05 mol NaOH, and a 10 µL aliquot were used for protein quantifica- determination, which were measured by enzyme immunoassay using an ELISA kit (Beijing tion. One gram protein of each sample was used for plasma and intestinal mucosa MT Sinouk Institute of Biological Technology, Beijing, China) analyzed with ELISA analyzer and 5-HTP determination, which were measured by enzyme immunoassay using an (Waldron dr-200 Bs) following the manufacturer’s instructions. ELISA kit (Beijing Sinouk Institute of Biological Technology, Beijing, China) analyzed with ELISA analyzer (Waldron dr-200 Bs) following the manufacturer’s instructions.

4.4. Statistical Analyses All data were analyzed using General Linear Model Procedure of repeated-measurement ANOVA with SAS statistical software (SAS 9.4, SAS Institute, Cary, NC, USA). Different doses of 5-HTP, different vessels of 5-HTP and MT concentration, and each time points of 5-HTP and MT concentration were analyzed separately with the model of Y = D + V + T + D * V + D * T + D * V * T. Here, D represents dosage, V represents vessels, T represents time. The least square means (LSM) and standard error of the mean

Molecules 2021, 26, 5275 11 of 13

4.4. Statistical Analyses All data were analyzed using General Linear Model Procedure of repeated-measurement ANOVA with SAS statistical software (SAS 9.4, SAS Institute, Cary, NC, USA). Different doses of 5-HTP, different vessels of 5-HTP and MT concentration, and each time points of 5-HTP and MT concentration were analyzed separately with the model of Y = D + V + T + D * V + D * T + D * V * T. Here, D represents dosage, V represents vessels, T represents time. The least square means (LSM) and standard error of the mean (SEM) were obtained from the model, the multiple comparisons with LSM were performed with PDIFF option. All data were expressed as LSM ± SEM. The statistical significance was set up p < 0.05, highly significant p < 0.01.

Author Contributions: Writing—original draft preparation and investigation, J.P.; Conceptualization and methodology, K.Y., G.L. and F.L.; Investigation and analysis, C.W. and X.L.; Software and data curation, S.Z. and W.Z.; Animal model and sample supplement, J.P., G.Z. and C.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was financed by the National Natural Science Foundation of China (grant number, 31760681) and, the Xinjiang Uygur Autonomous Region Graduate Innovation Project (grant number, XJ2020G135). Institutional Review Board Statement: All experimental procedures involving animals were approved (animal protocol number: 2020022) by the Animal Welfare and Ethics Committee of Xinjiang Agricultural University, Urumqi, Xinjiang, China. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in this article. Acknowledgments: The authors thank the Xinjiang key Laboratory of herbivore Nutrition for Meat and Milk production, and College of Veterinary Medicine of Xinjiang Agricultural University. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Samples of the compounds are not available from the authors.

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