Soybean protein hydrolysate stimulated cholecystokinin secretion and inhibited feed intake through calcium-sensing receptors and intracellular calcium signalling in pigs†

Lvyang Wang, a Liren Ding,b Weiyun Zhua and Suqin Hang*a

Although soybean protein is the major component in livestock feeds, its effect on pigs’ appetites is largely unknown. Recently, the importance of gut nutrient-sensing for appetite modulation by regulating anorec-
tic gut hormone release has been recognised. This study investigates the roles of soybean proteins in appetite regulation, anorectic gut hormone secretion, and underlying mechanisms. The duodenal-cannu- lated piglets were used to evaluate the effects of soybean protein hydrolysate (SPH) on feed intake and
anorectic hormone release, including cholecystokinin (CCK), peptide YY (PYY), glucagon-like peptide 1
(GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) in the hepatic vein by infusing SPH. Identifying which nutrient-sensing receptor in pig duodenum response to SPH stimulation for gut hormone release was conducted. Using its antagonist, the role of the identified receptor in feed intake
and anorectic hormone release was also investigated. Combination with an ex vivo perfusion system, the
possible mechanism by which SPH exerts the effects in porcine duodenum was further illustrated. Results
in vivo showed that intraduodenal infusion of SPH inhibited short-term feed intake in pigs and promoted CCK, PYY, and GIP secretion in the hepatic vein. SPH also increased duodenum calcium-sensing receptor (CaSR) expression. Pre-treated with CaSR antagonist NPS 2143, the feed intake of pigs tended to be atte- nuated by SPH (P = 0.09), and CCK release was also suppressed (P < 0.05), indicating that CaSR was involved in SPH-stimulated CCK release and inhibited feed intake in pigs. The ex vivo perfused duodenum tissues revealed that SPH-triggered CCK secretion was likeliest due to the activation of the intracellular Ca2+/TRPM5 pathway. Overall, this study’s result illustrates that the diet soybean protein might decrease appetite in pigs by triggering duodenum CCK secretion by activating CaSR and the intracellular Ca2+/ TRPM5 pathway.
1. Introduction
Numerous studies have indicated that protein is the macronu- trient with the highest satiating effect compared to an iso- energetic intake of fat and carbohydrates.1,2 Ingested protein evokes satiety signals attributed to its robust effects on stimu- lating anorectic hormone release from gut enteroendocrine cells (EECs), including cholecystokinin (CCK), peptide YY

(PYY), glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP).3,4 As an important source of protein in the livestock feeds, soybean proteins induce satiety with elevated CCK secretion in rats by gastric administration
before a meal.5 β-Conglycinin, a major component of soybean
protein, is effective to enhance fullness and reduce hunger sensations in healthy humans.6 However, the effects of soybean protein on pigs’ appetite and the anorectic hormone release associated with the feed intake evoked by soybean

protein remain partially unclear.

aNational Center for International Research on Animal Gut Nutrition, Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health, Laboratory of Gastrointestinal Microbiology, Nanjing Agricultural University, Nanjing 210095, China. E-mail: [email protected]; Fax: +86-25-84395314;
Tel: +86-25-84395037
bNational Experimental Teaching Center for Animal Science, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/ d1fo01596f

EECs are sparsely populated throughout the gastrointesti- nal tract (GIT) mucosa and thus directly in contact with ingested protein and its degradation products, mainly as pep- tides and amino acids.7,8 Several G protein-coupled receptors (GPCRs) expressed on the apical transporter membrane of EECs contribute to the protein-sensing mechanisms.9,10 One such candidate is the calcium-sensing receptor (CaSR), which can recognise various dietary peptides and L-amino acids,
thereby stimulating CCK, GLP-1, and PYY release.11–13 Also, several other GPCRs, including GPR93, GPRC6A, and umami taste receptor (T1R1/T1R3), have also been reported to regulate the secretion of CCK induced by protein hydrolysate and amino acids in vitro.14–16 Besides GPCRs, the potential role of an intestinal transporter peptide transporter 1 (PepT1) in casein stimulation of GLP-1 release has been recently con- firmed in rodents.17 This suggests that the mechanisms under- lying protein-induced anorectic hormone secretion are complex, multifactorial, and even contradictory, and largely carried out in single-cell lines and rodents. Besides, the expression pattern of nutrient-sensing related genes differed between the species (humans, pigs, and mice) in the proximal intestine, suggesting that the extrapolation of nutrient-sensing from one species to the other may be required with respect.18 More investigations are required in pigs.
Most GPCRs interact with heterotrimeric G proteins to initiate intracellular signalling cascades, thereby modulating various physiological process.19 Intracellular Ca2+ is recognised as an important regulator of GPCR-mediated signalling path- ways. Emerging evidence has shown an increasing intracellular Ca2+ concentration in response to the activation of several membrane in GPCRs.20 Furthermore, the CCK release in response to phenylalanine in STC-1 cells was Ca2+ depen- dent.21 This raised the hypothesis that intracellular Ca2+ might play a potential role in the GPCR-mediated protein-sensing mechanism.
This study assesses the effects of soybean protein hydroly- sate (SPH) on appetite and anorectic gut hormone secretion in piglets to identify which sensory receptors are involved in this protein-sensing and uncover the underlying molecular signal- ling mechanisms.
2. Materials and methods
2.1 Chemicals
Soybean protein isolate (SPI, protein content 90%) was pur- chased from Yuanye Biotechnology Co., Ltd, (Shanghai, China). Defatted soybean flour ( protein content 75%) was obtained from Zhaoyuan Wenji Food Co., Ltd, (Shandong, China). NPS 2143, cinacalcet, 2-aminoethyl diphenylborinate (2-APB), nifedipine, and EGTA were obtained from MedChem Express (Shanghai, China). Calhex 231 was purchased from ApexBio (Houston, TX, USA). Sodium bisulphite (SBS), tri- phenylphophine oxide (TPPO), and 2-hydroxypropyl-
β-cyclodextrin were obtained from Sigma-Aldrich (St Louis,
MO, USA). BAPTA-AM was obtained from Selleck (Houston, TX, USA).

2.2 Protein isolation, identification, and hydrolysate
2.2.1 β-Conglycinin (7S) and glycinin (11S) isolation. The two major storage proteins in soybeans are β-conglycinin (7S) and glycinin (11S). They were obtained following a previous
method with slight modifications.22 Briefly, 5 g defatted soybean flour was dissolved in 75 ml of Tris-HCl ( pH 8.5) (w/v

= 1 : 15) and homogenised, stirring at 120 rpm, 45 °C for 2 h, and then centrifuged at 9000g, 4 °C for 30 min. The super- natant supplemented with solid SBS to a final concentration of
0.01 M, adjusted to pH 6.4 using 2 N HCl, and the solution was kept at 4 °C overnight, which was then centrifuged at 6500g, 4 °C for 30 min. To obtain the 11S fraction, the precipi- tate was washed three times using pure water and dissolved in
0.03 M Tris-HCl, adjusted to pH 7.0 using 2 N NaOH, frozen at
−20 °C, and then lyophilised in a FreeZone 4.5 L Freeze Dry System (Labconco Corp., Kansas City, MO, USA). To obtain the 7S fraction, the supernatant supplemented with solid NaCl
was added to a final concentration of 0.25 M, adjusted to pH
5.5 using 2 N HCl, and then stirred for 0.5 h at room tempera- ture. The solution was centrifuged at 9000g, 4 °C for 30 min. The supernatant was diluted two-fold with pure water, adjusted to pH 4.8 using 2 N HCl, and then centrifuged at 6500g, 4 °C for 20 min. The precipitate was dissolved in 0.03 M
Tris-HCl, adjusted to pH 7.0, frozen at −20 °C, and then
lyophilised.
2.2.2 Hydrolysate preparation. Soybean protein hydrolysate (SPH), β-conglycinin hydrolysate (7SH), and glycinin hydroly- sate (11SH) were prepared following the previous study with
slight modifications.23 Briefly, SPI/7S/11S was suspended in distilled H2O (10%, w/v) and adjusted to pH 2.0 using 2 N HCl. Then, the porcine pepsin was added to hydrolyse SPI/7S/11S at a ratio 1 : 100 (w/w) of enzyme (Yuanye Biotechnology Co., Ltd, Shanghai, China). After 1 h of constant agitation at 37 °C, the reaction was terminated by increasing the solution pH to 7.0 with 2 N NaOH. The obtained hydrolysates were frozen at
−20 °C, and lyophilised for further use.
2.2.3 SDS-polyacrylamide gel electrophoresis. To deter- mine the effectiveness of hydrolysis and the isolation of 7S and 11S, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted following a previous method.22 Briefly,
samples (1 μg μl−1) were mixed with 5× SDS-PAGE loading
buffer (Beyotime, Shanghai, China) and then heated in a boiling water bath for 5 min. Ten microliter denatured protein samples were loaded on a 5% stacking gel and frac- tionated on a 12% separating gel, which was made using an SDS gel preparation kit (Beyotime, Shanghai, China). A pre- stained colour protein marker (Beyotime, Shanghai, China) was used for determining molecular weight. The separating gel was stained in a commassie blue staining solution (Biosharp, Nanjing, China) and destained using a commassie blue staining destaining solution (Biosharp). The gel images were digitised using a Tanon 5200Multi imaging system (Tanon Science & Technology Co., Ltd, Shanghai, China) and analysed using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).
2.3 Animal surgery
Eighteen castrated piglets (Duroc × Landrace × Large White) at an average age of 50 days with an initial weight of 14.5 ± 0.2 kg were purchased from a local commercial pig farm in Nanjing, China. All piglets were housed in individual metabolic cages with a controlled temperature of 25 ± 2 °C and had free access
to commercial piglet feeds provided by Huizhou Aohua Feeds Co., Ltd (Huizhou, China). The basal diet was meet the nutri- ent requirements of the pigs according to the National Research Council (NRC) (2012) standards (Table S1†). After one-week acclimation, each piglet was surgically fitted with a simple T-cannula (8.2 cm length, 10 cm width, and 1.5 cm internal diameter) in the duodenum ( just posterior to the pan- creatic and bile duct) for saline or SPH infusion, and a catheter in the hepatic vein for blood sampling following previous studies.24,25 To avoid potential infections, all the cannulated piglets were intramuscularly injected with ceftriaxone sodium and treated with iodine tincture in the wound and adjacent skin for one week (twice a day). To prevent the formation of blood clots, the catheters were filled and flushed with aseptic saline solution (200 IU heparin per mL) twice daily. The formal trials were not started until the feed intake of pigs was stabilised (about 15 d after surgery). Fig. 1A shows the general experiment timeline.
2.4 Experimental design in vivo studies
The in vivo experiments comprised two short-term trials (trial 1 and trial 2) using the same batch of piglets. Each trial was performed on three consecutive test days. After the trial 1, piglets had free access to feedstuffs and water for 7 days (a

recovery period) till the trial 2. Fig. 1B (trial 1) and C (trial 2) present the experimental design and timeline.
Experiment 1: After recovering from T-cannula surgery, 12 piglets were randomly divided into the control group (saline infusion, CON) and SPH infusion group (SPH) (n = 6 per group) with no difference in body weight (20.1 ± 0.3 kg) and feed intake. After 12 h of fasting, piglets were given an intra- duodenal infusion of 15 mL saline (CON) or SPH solution (SPH) at 10 min intervals from 8:00 to 8:50 in the morning. SPH solution was prepared by dissolving 20 g SPH in 90 mL saline according to an infusion dose of 1 g per kg body weight and then adjusted to the pH of 5.0, which is about the native pH of porcine duodenum.26 At the end of infusion (at 9:00), 1 mL of blood samples from the hepatic vein were collected using sterile syringes and placed in ice-chilled heparinised vacuum tubes (Jiangsu Kangjie Medical Devices Co, Ltd, Jiangyan, China). Duodenal mucosal samples were obtained and stored in liquid nitrogen for further analysis using dispo- sable biopsy forceps (Yangzhou Fuda Medical Devices, Co., Ltd, Yangzhou, China). Then, the pigs were provided an excess amount of feed (700 g) at 9:00. The residual feeds per cage were recorded at 10:00, 11:00, and 13:00 to calculate the feed consumption of each piglet after 1, 2, and 4 h of feeding, respectively (Fig. 1B). The blood samples were centrifuged at

 

Fig. 1 The timeline of in vivo experiments. (A) Eighteen piglets were operated on with a duodenal cannula. After 15 d recovery (including initial 7 d with antibiotic treatment), trial 1 was conducted, and then after 7 d recovery, trial 2 was conducted. (B) Experiment design of the experiment 1. Twelve piglets were randomly assigned to the control (CON) and SPH groups (n = 6 per group). After 12 h of fasting, the CON and SPH were duoden- ally infused with 15 mL saline or 1.00 g kg−1 SPH at 10 min intervals from 8:00–8:50 in the morning. The hepatic vein blood and duodenal mucosa
were collected at 9:00, and short-term feed intake at 1, 2, and 4 h were measured. (C) Experiment design of the experiment 2. Eighteen piglets were
randomly assigned to the control (CON), SPH, and SPH + NPS 2143 groups (n = 6 per group). The CON and SPH were both intraduodenally infused with 15 ml saline at 7:45 in the morning, while the SPH + NPS 2143 was infused with 15 ml of NPS 2143 solution (25 μM). From 8:00–8:50, CON, SPH, and SPH + NPS 2143 were duodenally infused with saline and SPH at 10 min intervals, respectively. The hepatic vein blood was collected
during 7:45–9:00 at 15 min intervals, and short-term feed intake at 1, 2, and 4 h were measured.
3000g, 4 °C for 10 min to separate plasma, and then the plasma samples were stored at −80 °C for further analysis of anorectic hormones.
Experiment 2: To evaluate whether CaSR was involved in the decrease in feed intake induced by SPH and anorectic hormone release, the CaSR antagonist NPS 2143 was used. Eighteen piglets were assigned to three groups at random (n = 6 per group): the control group (CON), SPH treatment group (SPH), and SPH + NPS 2143 treatment group (SPH + NPS 2143). At 7:45 in the morning, piglets in CON and SPH with 12 h of fasting were intraduodenally infused with 15 ml saline, while piglets in SPH + NPS 2143 were infused with 15 ml NPS 2143
solution (25 μM). To aid solubility, NPS 2143 was dissolved in saline with 20% 2-hydroxypropyl-β-cyclodextrin.27 From 8:00 to
8:50, piglets in CON, SPH, or SPH + NPS 2143 were respectively given an intraduodenal infusion of 15 mL either saline or SPH at 10 min intervals. One millilitre of blood sample from the hepatic vein was collected into ice-chilled heparinised vacuum tubes every 15 minutes from 7:45 using sterile syringes. At the end of infusion (at 9:00), the piglets were provided an excess amount of feeds (700 g). The residual feed per cage was recorded at 10:00, 11:00, and 13:00 to calculate the feed con- sumption of each piglet after 1, 2, and 4 h of feeding, respect- ively (Fig. 1C). The blood samples were centrifuged at 3000g,
4 °C for 10 min to separate plasma, and then the plasma samples were stored at −80 °C for further analysis of anorectic hormones.

2.5 Perfusion experiments (ex vivo studies)
2.5.1 Porcine duodenum collection and sample prepa- ration. The fresh porcine duodenum was obtained from three pigs (Duroc × Landrace × Large White) killed for meat pro- duction at a local abattoir. The pigs were aged around six months and weighed about 100 to 130 kg. Within 15 min after slaughter, the entire duodenum was immediately excised and transported in ice-cold oxygenated (95% O2, 5% CO2) Krebs’ ringer bicarbonate buffer (KHB) to the laboratory for sample preparation. The KHB contained 120 mM NaCl,
4.5 mM KCl, 25 mM NaHCO3, 20 mM Hepes, 1.8 mM CaCl2,
0.5 mM MgCl2, and 10 mM D-glucose ( pH = 7.4). Upon reaching the laboratory, approximately 5 cm segments of the porcine duodenum after the pylorus were longitudinally cut open and gently rinsed with 0.01 M phosphate-buffered saline (PBS, Sigma-Aldrich, St Louis, MO, USA) to remove intestinal contents, and the outer muscle layers were care- fully stripped off. The mucosal tissues were then cut into small slices (approximately 1 mm2) and mixed thoroughly. All of the above procedures were conducted under sterile con- ditions. Then, mixed tissues (400 mg) were randomly placed into chambers for the perfusion experiment (n = 6–8 per group). The perfusion system was previously established by Zhao et al.28
2.5.2 Ex vivo perfusion. Experiment 3: To investigate CCK release in response to SPH and SPI, the mucosal tissues were perfused with different concentrations of SPH/SPI for 120 (Fig. 2A and its legend).

Experiment 4: To identify the role of CaSR in SPH-stimu- lated CCK release, the mucosal tissues were respectively per- fused with SPH in addition to CaSR antagonist NPS 2143 (25 μM)/Calhex 231(20 μM), and CaSR agonist cinacalcet
(1 μM) (Fig. 2B and its legend).
Experiment 5: To demonstrate the role of intracellular Ca2+ in SPH-induced CCK secretion, mucosal tissues were perfused
with SPH in addition to intracellular Ca2+ ([Ca2+]i) chelator BAPTA-AM (25 μM), IP3R inhibitor 2-APB (50 μM), extracellular Ca2+ ([Ca2+]e)chelator EGTA (1 mM), and L-type voltage-gated calcium channels (VGCC) blocker nifedipine (1 μM), respect- ively (Fig. 2C and its legend).
Experiment 6: To evaluate the role of transient receptor potential channel type M5 (TRPM5) and cell depolarisation in SPH-induced CCK release, the mucosal tissues were perfused with SPH in addition to TRPM5 blocker TPPO, KATP channel
opener diazoxide (500 μM), and removal of extracellular Na+,
respectively (Fig. 2D and its legend). The procedure for KCl stimulation was a slight difference from the above treatments, which the mucosal tissues were perfused with KHB at 0–30 min, and then with KCl at 30–60 min.
Experiment 7: To identify the effects of two dominant protein compounds, 7S and 11S, in SPI on CCK secretion, the mucosal tissues were perfused with 1% 7SH/11SH/SPH, respectively, and Calhex 231 was applied to demonstrate the mediation role of CaSR (Fig. 2E and its legend).
SPH and chemicals for perfusion were diluted in KHB solu- tion ahead. As mucosal tissues were placed in the chambers, perfusion was started to equilibrate with KHB for 30 min at a rate of 0.1 ml min−1. During perfusion, after each stimulation,
the mucosal tissues were immediately flushed with the
exchanged solution at a flow rate of 2 ml min−1 for 2 min to replace the residual perfusate in the chambers and silicon
tubes (the total volume is less than 4 ml), thereafter at 0.1 ml min−1 until the end of this stimulation. For better dissolving, NPS 2143, Calhex 231, cinacalcet, nifedipine, 2-APB, and
BAPTA-AM were dissolved in DMSO, while TPPO was diluted in ethanol, and then they were further diluted in KHB buffer until the final concentration of DMSO or ethanol was less than 0.1% (v/v) to keep the duodenal tissue viability. The perfusate was collected every 10 min, and then centrifuged at 10 000g for 5 min at 4 °C. The supernatant was retrieved and stored at
−80 °C for CCK and LDH analysis.
2.6 Determination of hormone concentration and LDH activity
The hormone concentrations were evaluated using pig com- mercial ELISA kits CCK (ANG-E31022P), PYY (ANG-E31368P), GIP (ANG-E31000P), and GLP-1 (ANG-E311182P) (Angle Gene
Biotechnology Co., Ltd, Nanjing, China) following the manu- facturers’ instructions. The sensitivity for detecting CCK was
10 ng L−1 (intra- and inter-assay variation coefficients were
<9% and <15%, respectively); for detecting PYY was 4 ng L−1
(intra- and inter-assay variation coefficients were <9% and
<12%, respectively); for detecting GLP-1 was 2 ng L−1 (intra- and inter-assay variation coefficients were <9% and <11%,

 

Fig. 2 The timeline of ex vivo experiments. (A) The scheme of the experiment 3. After 30 min equilibrium with KHB, the mucosal tissues were per- fused with KHB from 0–30 min (control), then, KHB supplemented with SPH/SPI at a final concentration of 0.1% (w/v) from 30–60 min, 1% (w/v) from 60–90 min, and 5% (w/v) from 90–120 min. The perfusate was collected every 10 min to determine the CCK concentration. (B–D) Scheme of
the experiment 4–6. After 30 min equilibrium with KHB, the mucosal tissues were perfused with KHB from 0–20 min, 1% SPH (w/v) from 20–50 min, KHB + antagnoists/agonists from 50–70 min, and 1% SPH (w/v) + antagonists/agonists from 70–100 min. The perfusate was collected every 10 min to determine the CCK concentrations. (E) The scheme of the experiment 7. After 30 min equilibrium with KHB, the mucosal tissues were perfused with KHB from 0–20 min, 1% 7SH/11SH/SPH (w/v) from 20–50 min, KHB + CaSR antagonist from 50–70 min, and 1% SPH (w/v) + CaSR antagonist from 70–100 min. The perfusate was collected every 10 min to determine the CCK concentrations.

respectively); and for detecting GIP was 2 ng L−1 (intra- and inter-assay variation coefficients were <9% and <11%, respect-
ively). The measurement of LDH in the perfusate was per- formed using an LDH assay kit (A020-2-2) (Nanjing Jiancheng Bioengineering Institution, Nanjing, China) following the manufacturer’s instrument manual. Relative LDH release (%) was calculated as after exposure to chemicals (at 70 min) vs. KHB (at 20 min, as 100%).

2.7 RNA extraction and real-time quantitative PCR
Total RNA from the porcine duodenal mucosa was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The quantity and purity of RNA were analysed by measuring the absorbance at 260 and
280 nm using NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MhgA, USA). cDNA synthesis was per- formed using PrimeScriptTM RT Master Mix (TaKaRa, Dalian, China). Triplicate qRT-PCR was performed using a QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with TB Green® Fast qPCR Mix (Takara, Dalian, China) following the manufacturer’s instructions. All the oligonucleotide primers were purchased from Invitrogen (Shanghai, China), and Table S2† presents the

primers sequences. The ΔΔCt method was used to calculate the relative gene expression, where each sample was normal- ised to β-actin as the reference gene.
2.8 Statistical analysis
Data were plotted for individual animals with group means and the standard error of the mean (SEM). Statistical analyses were performed using IBM SPSS Statistics 25. Comparisons of the two groups in vivo study were analysed with unpaired Student’s t-test. The datasets from ex vivo perfusion were expressed as averaged concentrations during each treatment and then analysed with a paired Student’s t-test. One-way ana- lysis of variance (ANOVA) was conducted for more than two groups, followed by Tukey post hoc test. Significant differences were considered at P < 0.05.

3. Results
3.1 In vivo studies
3.1.1 Acute administration of SPH inhibited feed intake and increased anorectic gut hormone secretion. SDS-PAGE profiles of SPH (Fig. 3A) present a reduction in molecular weight after 1 h treatment with porcine pepsin, suggesting that

 

Fig. 3 Effects of SPH on short-term feed intake and anorectic gut hormones release. (A) SDS-PAGE profile of SPH. (B) Short-term feed intake was recorded. (C–F) CCK, PYY, GIP, and GLP-1 concentrations in the hepatic vein were detected. (G) Correlation analysis between the 1 h feed intake and CCK level. Values are the means ± SEM (n = 6). *P < 0.05 (by unpaired Student’s t-test).

SPI was remarkably degraded. Fig. 3B shows that SPH infusion reduced the cumulative 1 h feed intake of pigs (P < 0.05) com- pared with CON, while it did not affect 2-and 4 h feed intake (P
> 0.05). The plasma CCK, PYY, and GIP levels in the hepatic vein were increased by SPH (P < 0.05) (Fig. 3C–E), while the GLP-1 level was not affected (P > 0.05) (Fig. 3F). Particularly, a significant inverse correlation was observed between the cumulative 1 h feed intake and plasma CCK level (Fig. 3G).
3.1.2 mRNA expression of duodenal chemosensing recep- tors. To identify luminal chemosensing receptors in response

 

Fig. 4 The mRNA expression of nutrient-sensing receptors and trans- porters in responses to SPH in porcine duodenum. The expression of CaSR, PepT1, GPR93, GPRC6A, T1R1, and T1R3 were detected. Values are the means ± SEM (n = 6). *P < 0.05 (by unpaired Student’s t-test).

to SPH, we investigated the mRNA expression of several poten- tial receptors in the porcine duodenum after treatment with SPH. Results showed that the SPH infusion increased the mRNA expression of CaSR and PepT1 (P < 0.05) (Fig. 4), whereas no differences were observed in GPRC6A, T1R1, T1R3, and GPR93 between SPH infusion and the control (P > 0.05), indicating that CaSR and PepT1 might play important roles in SPH-induced anorectic gut hormone secretion.
3.1.3 Effects of CaSR antagonist on feed intake and anorec- tic gut hormone secretion. To directly address whether CaSR is involved in SPH-inhibited feed intake and induced anorec- tic gut hormone secretion, the CaSR antagonist NPS 2143 was introduced into the duodenum. Observably, SPH decreased the cumulative 1 h feed intake of pigs (P < 0.05), while SPH + NPS 2143 resulted in a modest reduction of 1 h feed intake (P = 0.09) (Fig. 5A), suggesting that the anorectic effect of intraduodenal infusion of SPH was intended to attenuate in the presence of NPS 2143. The plasma CCK level in the hepatic vein was higher from 30 to 60 min with SPH infusion compared with CON (P < 0.05) (Fig. 5B), while PYY and GIP were only higher at 60 min with SPH infusion com- pared with CON (P < 0.05) (Fig. 5C and D). Importantly, in the presence of NPS 2143, CCK secretion induced by SPH was moderately decreasing compared to that in SPH from 30 to 45 min (P > 0.05) and was significantly reduced at 60 min compared with SPH (P < 0.05). The area under the curve (AUC) analysis also revealed that NPS 2143 administration considerably reduced the total level of CCK release compared with the SPH group (P < 0.05). In contrast, there was no sig-
Fig. 5 Effects of the CaSR antagonist on short-term feed intake and anorectic gut hormone release by SPH stimulation. (A) Short-term feed intake was recorded. (B–E) The kinetics CCK, PYY, GIP, and GLP-1 concentrations were detected in the hepatic vein (inset: integrated area under the curve [AUC]0–60 min). Values are the means ± SEM (n = 6). Different letters represent significant differences (P < 0.05), *P < 0.05 (by Tukey post hoc test).
nificant difference observed in PYY, GIP, and GLP-1 concen- trations between SPH and SPH + NPS 2143 during the infu- sion (P > 0.05) (Fig. 5C–E). Therefore, these results indicate that CaSR may participate in SPH-induced CCK release to inhibit feed intake in porcine.
3.2 Ex vivo studies
3.2.1 CaSR mediated SPH-induced CCK release from per- fused porcine duodenal mucosa. SPH at 1% and 5% dose- dependently increased CCK secretion from the duodenal mucosa (P < 0.05) (Fig. 6A and B). Although 5% SPI also stimu- lated CCK secretion, the CCK secretory capacity was weaker than that of SPH. Based on these results, we used a dose of 1% SPH in further experiments. Importantly, the CCK levels in response to two consecutive 1% SPH stimulations were both elevated compared to their respective baselines but no differ- ences from each other, which confirmed that the intestinal tissues were viable during 100 min perfusion (P > 0.05) (Fig. 6C and D). The level of LDH activity in the perfusate did not change after exposure to chemicals (e.g. NPS 2143),

suggesting that treatments such as NPS 2143 and Calhex 231 did not damage the viability of the intestinal tissue (Fig. S1†).
Consistent with the in vivo results, the CCK response to 1% SPH was attenuated by treatment with the CaSR antagonist, NPS 2143, or Calhex 231 (P < 0.05) (Fig. 7A, B and D, E). In con- trast, perfusion of the mucosa tissue with CaSR agonist cina- calcet increased the CCK release (P < 0.05) (Fig. 7C and F). PepT1 inhibitor 4-AMBA and substrate cephalexin had no effects on CCK levels by 1% SPH stimulation (P > 0.05) (Fig. S2A–D†), indicating that PepT1 was not involved in CCK secretion in the porcine duodenum. Therefore, these results demonstrate that CaSR plays a critical role in SPH-induced CCK secretion from the porcine duodenum.
3.2.2 SPH-stimulated CCK secretion relies on intracellular Ca2+ signalling. Fig. 8 summarises the potential role of intra- cellular Ca2+ in SPH-induced CCK release using selective antagonists. It revealed that the CCK secretion response to 1% SPH was abolished after perfusion with intracellular Ca2+ ([Ca2+]i) chelator BAPTA-AM (P < 0.05), suggesting that the presence of free [Ca2+]i is required for SPH-stimulated CCK

 

Fig. 6 SPH stimulates CCK release from porcine duodenal tissues. (A and B) The mucosal tissues were perfused with 0% (KHB, from 0–30 min), 0.1% from 30–60 min, 1% from 60–90 min, and 5% from 90–120 min SPH/SPI for a total 120 min. The perfusate was obtained every 10 min to deter- mine the CCK concentrations. (C and D) The mucosal tissues were perfused with KHB from 0–20 min (baseline), 1% SPH (20–50 min), KHB from 50–70 min (baseline), and 1% SPH from 70–100 min. The perfusate was collected every 10 min to determine the CCK concentrations. Values are the
means ± SEM (n = 7). “*” represents a significant difference between baseline and SPH stimulant (*P < 0.05), “#” means a significant difference
between SPH stimulants (#P < 0.05 (by paired Student’s t-test).
Fig. 7 CaSR mediated SPH-induced CCK release from porcine duodenal tissues. (A–F) The mucosal tissues were perfused with KHB from 0–20 min (baseline), 1% SPH from 20–50 min, KHB + NPS 2143/Calhex 231/cinacalcet from 50–70 min (baseline), and 1% SPH + NPS 2143/Calhex 231/cinacal- cet from 70–100 min. The perfusate was collected every 10 min to determine the CCK concentrations. Values are the means ± SEM (n = 6–8). “*” represents a significant difference between baseline and SPH stimulant (*P < 0.05), “#” means a significant difference between SPH stimulants (#P <
0.05) (by paired Student’s t-test).

secretion (Fig. 8A and E). Similarly, IP3R inhibitor 2-APB, VGCC blocker nifedipine, and the absence of extracellular Ca2+ ([Ca2+]e) all attenuated the CCK response to 1% SPH stimulation (P < 0.05) (Fig. 8B–D and F–H). Therefore, our data show that SPH-induced CCK secretion is dependent on Ca2+.
3.2.3 SPH-stimulated CCK release depends on TRPM5 and cell depolarisation. Fig. 9 shows the role of TRPM5 and sub- sequent cell depolarisation in CCK secretion triggered by SPH.

Treatment with TRPM5 blocker TPPO decreased the CCK release response to 1% SPH stimulation (P < 0.05) (Fig. 9A and E). When extracellular Na+ ion was substituted with non-per- meable ion NMDG+, the effects of SPH on CCK release were reduced (P < 0.05) (Fig. 9B and F). Furthermore, KCl-induced depolarisation potentiated CCK release, while KATP channel opener diazoxide, which resulted in hyperpolarisation, attenu- ated the CCK secretion in response to 1% SPH (P < 0.05) (Fig. 9C, D and G, H). It indicates that SPH-induced CCK

Fig. 8 SPH stimulates CCK release in the porcine duodenum, depending on intracellular Ca2+. (A–H) The mucosal tissues were perfused with KHB from 0–20 min (baseline), 1% SPH from 20–50 min, KHB + BAPTA-AM/2-APB/nifedipine/EGTA from 50–70 min (baseline), and 1% SPH +
BAPTA-AM/2-APB/nifedipine/EGTA from 70–100 min. The perfusate was collected every 10 min to determine the CCK concentrations. Values are the means ± SEM (n = 6–8). “*” represents a significant difference between baseline and SPH stimulant (*P < 0.05), “#” means a significant difference between SPH stimulants (#P < 0.05) (by paired Student’s t-test).

Fig. 9 The involvement of TRPM5 and cell depolarisation in SPH-stimulated CCK release. (A–C and E–G) The mucosal tissues were perfused with KHB from 0–20 min (baseline), 1% SPH from 20–50 min, KHB + TPPO/NMDG+/diazoxide from 50–70 min (baseline), and 1% SPH + TPPO/NMDG+/ diazoxide from 70–100 min. The perfusate was collected every 10 min to determine the CCK concentrations. (D and H) The mucosal tissues were perfused with KHB from 0–30 min and KCl from 30–60 min. The perfusate was collected every 10 min to determine the CCK concentrations.
Values are the means ± SEM (n = 6–8). “*” represents a significant difference between baseline and SPH stimulant (*P < 0.05), “#” means a significant difference between SPH stimulants (#P < 0.05) (by paired Student’s t-test)

release requires TRPM5 activation and Na+ influx, thereby inducing cell depolarisation.
3.2.4 β-Conglycinin (7S) is the major component of CCK
secretion. To identify the dominant compound in SPH respon- sible for inducing CCK secretion, the two major protein com-

ponents of SPI, 7S and 11S, were isolated, and the effects of their hydrolysates on CCK release were investigated, respect- ively. SDS-PAGE profiles show that 7S and 11S were success- fully isolated from defatted soybean flour (Fig. 10A). After hydrolysis, the effects on CCK release were in the rank order of

Fig. 10 Effects of 7SH and 11SH on CCK release in response to SPH. (A) Identification of the isolated 7S and 11S by SDS-PAGE. M: marker. (B and C) The mucosal tissues were perfused with KHB from 0–20 min, 1% 7SH/11SH/SPH from 20–50 min, KHB + Calhex 231 from 50–70 min, and 1% 7SH/ 11SH/SPH + Calhex 231 from 70–100 min. The perfusate was collected every 10 min to determine the CCK concentrations. Different letters indicate significant differences (P < 0.05 by Tukey post hoc test).

potency of 7SH > SPH > 11SH, suggesting 7SH was the major component in SPH-induced CCK secretion (Fig. 10B and C). Similarly, when the CaSR inhibitor Calhex231 was added, CCK secretion was significantly suppressed

4. Discussion
Dietary nutrients reach the small intestine through a series of physiological processes, including chewing, swallowing, and gastric emptying. To exclude the potential influence of these behaviours in appetite and gut hormone release, a duodenal- cannulated pig model was used in this study, which allowed the nutrients to directly act on the small intestine. Then, soybean protein hydrolysate was chosen as the dietary com- ponent, which mimics the dietary proteins digest in the luminal chyme. Our results indicated that duodenal infusion of SPH inhibits feed intake in pigs, agreeing with several studies of other protein hydrolysates, including casein, whey, potato, and wheat gluten in humans or rodents appetite.29–32 Also, consistent with previous studies that described dietary protein as a potent stimulant of anorectic gut hormone release, our study also demonstrated that SPH enhanced CCK, PYY, and GIP secretion. Importantly, an inverse correlation between feed intake and the plasma CCK level was observed. As an anorectic hormone, CCK is released from enteroendo- crine I-cells located in the upper small intestine.33 Zhang reported that active immunisation against CCK8 improved the feed intake and growth performance of pigs.34 Therefore, it is reasonable to suggest that SPH-induced appetite inhibition strongly depends on CCK release. However, we did not find any significant change in GLP-1 concentration after treatment with SPH, which was seemingly at odds with several reports

observing increased GLP-1 levels after intraduodenal infusion of whey protein hydrolysate.30 The reasons for this discrepancy are unknown, but it might be associated with composition differences between proteins because it has been demon- strated that preloads with equivalent casein and whey in healthy subjects could exert different effects on gut hormone secretion.35
It is recognised that dietary protein reached the duodenum in a partially digested form, which comprises large numbers of polypeptides and negligible amounts of free amino acids;36 even around 70% of protein nitrogen is found as peptides at distal locations ( jejunum).4 Consistent with this, the mRNA expression of GPRC6A and T1R1/T1R3, amino acid sensing receptors, was not affected after SPH treatment in this study. SPH did not affect the transcript level of GPR93, a peptide- sensing receptor, which overexpression amplifies STC-1 cells CCK transcription and secretion in response to meat protein hydrolysate.14 It is likeliest that the inherent limitation of the cell culture, which loses the interactions with adjacent cells (i.e. epithelial, goblet, paneth, and neuronal), occurs with EEC in vivo.
Intraduodenal infusion of SPH enhanced the mRNA expression of CaSR and PepT1. Our previous study confirmed that CaSR was expressed at a protein level in the porcine duo- denum.28 Although several studies have demonstrated that CaSR is crucial for various protein hydrolysate-induced CCK secretion in vitro,11,37 it is unknown whether this sensing mechanism remains under physiological conditions. A recent study identified the role of CaSR in the regulation of GLP-1 release through the intraileal infusion of a CaSR antagonist in cannulated rats.12 Following this way, by intraduodenal infus- ing of CaSR antagonist NPS 2143, there was a trend towards lowering the feed intake and the total CCK release compared
with the SPH group. The CCK levels were gradually increased from 30 min, which is similar to the findings revealed by Cuber et al.38 They found that growing pigs were intraduoden- ally infused with casein hydrolysate increased the plasma CCK during the first 30 min. Notably, the NPS 2143 reversed CCK secretion in response to SPH at 60 min, indicating that the inhibition of CaSR reduces the CCK release induced by SPH. The NPS 2143 treatments had no obvious effects on PYY and GIP secretion. Although previous studies have reported that PYY and GIP release were mediated by CaSR in response to the amino acid,39,40 the secretion in response to protein hydroly- sate may involve other mechanisms that have not been widely studied up to now. Therefore, the data in our in vivo study suggest that activation of CaSR is involved in the SPH-stimu- lated CCK secretion to induce an anorectic response.
Using an ex vivo perfusion system, we drew a similar con- clusion with in vivo results that CaSR modulates the SPH- induced CCK secretion from porcine duodenal mucosa tissues. Previous studies have illustrated that CCK secretion was partially impaired in response to a casein hydrolysate
(tryptone) in primary I cells isolated from CaSR−/− mice.41
Another evidence showed that after removal of free amino acids, CaSR played a similar mediation role on CCK release with the azuki or β-conglycinin hydrolysate stimulation,
suggesting that large peptides are attributed to CaSR activation
to induce CCK secretion rather than free amino acids.11 Notably, though the mRNA expression of PepT1 was signifi- cantly increased after SPH treatment in vivo, our ex vivo results showed that the CCK release by SPH stimulation did not result from PepT1, supporting a previous study in isolated CCK-pro- ducing cells,42 suggesting that the upregulated expression of PepT1 controls the transport of emerging oligopeptides rather than exerting a secretory effect.
The role of Ca2+ signalling in CCK release has been exam- ined previously. In STC-1 cells, bombesin-caused CCK secretion was reduced by treatment with L-type voltage-gated Ca2+ channels (VGCCs) blockers, indicating that CCK secretion required an influx of extracellular Ca2+ into the cell through VGCCs.43 Similarly, STC-1 cell release CCK in response to deox- ynivalenol was suppressed by adding PLC inhibitor and IP3 receptor antagonist, indicating that PLC-dependent secretion of intracellular Ca2+ stores were involved in CCK release.44 Therefore, it was hypothesised that CCK secretion in response to SPH might also be calcium-dependent. Previous studies directly observed the intracellular Ca2+ mobilization in single cells by confocal Ca2+ imaging using the Ca2+-sensitive indi- cator dye or fluorochrome, but it is hard for the perfused tissue to be conducted by the same method in the current study. Thus, antagonists were used in our study to test the hypothesis. There are two potential pathways that contribute to the increase in intracellular Ca2+ levels: (1) IP3R-gated Ca2+ leak from the endoplasmic reticulum Ca2+ stores and (2) extra- cellular Ca2+ influx via membrane VGCCs. Our data indicate that the CCK responses to SPH stimulation were attenuated when blocking these pathways, confirming that SPH-induced CCK release depends on increased intracellular Ca2+.

Previous studies have established that IP3R-mediated release of Ca2+ stores directly activates a transient receptor potential channel type M5 (TRPM5), which is involved in the final step of a G-coupled signalling cascade that culminates in membrane depolarisation.45,47 Bhavik et al. reported that CCK secretion in STC-1 cells was significantly attenuated by blocking the TRPM5 channel through its inhibitor TPPO or by siRNA specific to TRPM5.46 We observed a lower CCK release in response to SPH upon application of TPPO in the perfusion. As a monovalent cation-permeable channel, TRPM5 activation mediated extra- cellular Na+ entry and caused depolarisation, which provided the stimulus required for VGCCs activation.45,47 Our findings demonstrated that the removal of extracellular Na+ or hyperpol- arisation, induced by treatment with KATP-channel opener diaz- oxide, decreased the SPH-induced CCK secretion, while cell depolarisation caused by KCl stimulated CCK release. It implies that Na+ entry is crucial for CCK release stimulated by SPH. Combining the role of Ca2+ in CCK secretion in response to SPH, it is reasonable to postulate that SPH initiates the mobilis- ation of intracellular Ca2+ stores opening the TRPM5, causing depolarisation for activating VGCCs. In turn, the opened VGCCs facilitated extracellular Ca2+ influx, which further elevated the intracellular Ca2+ and stimulated CCK secretion.
Finally, the 7S and 11S proteins were isolated, and their effects on CCK release were explored. Our results indicated that 7SH was the major component in SPH-induced CCK secretion, supported by Nishi et al., who found that intraduo-
denal administration of 7S peptone suppressed food intake and increased the plasma CCK in rats.48 The β51-63 fragment enriched with arginine residues is the bioactive appetite
involved in 7S that triggers this effect.49

5. Conclusion
This study demonstrated that intraduodenal infusion of SPH suppressed feed intake and stimulated CCK secretion mediated by CaSR in piglets. Further, the CCK secretory mechanisms were likeliest due to activation of the intracellular Ca2+/TRPM5 pathway in the porcine duodenal tissue using an ex vivo per- fusion system. Our findings will be useful for understanding the feeding behaviour of pigs and provide new framework guidelines for strategies for manipulating feed intake in pigs.

Ethics approval
All animal procedures in this study were consistent with the Experimental Animal Care and Use Guidelines of China (Chinese Science and Technology Committee, 1988), and approved by the Animal Care and Use Committee of Nanjing Agricultural University (SYXK-2017-0027).

Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
This research was funded by the National Key Basic Research Program of China (2013CB127301)

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