# Methionine-Mediated Regulation of Intestinal Structure and Lipid Transport in the Rice Field Eel (Monopterus albus)

Authors: Yajun Hu; Minglang Cai; Huan Zhong; Wuying Chu; Yi Hu
Journal: Aquaculture Nutrition
 (2022)

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## Abstract

An eight-week feeding trial discusses how methionine affects intestinal barrier and lipid transport on rice field eel (Monopterus albus). Six isoenergetic and isonitrogenous feeds contained different levels of methionine (0, 2 g/kg, 4 g/kg, 6 g/kg, 8 g/kg, or 10 g/kg). Compared with M0 (0 g/kg), gastric amylase, lipase, and trypsin were remarkably increased as dietary methionine (P<0.05); intestinal amylase, lipase, and trypsin were remarkably increased in M8 (8 g/kg) (P<0.05). Compared with M0, gastric fovea was remarkably increased (P<0.001), gastric epithelium is neater in M8 than that in M0, intestinal villus height and muscular thickness are remarkably increased in M8 (P<0.001), and amounts of goblet cells per root in M8 were increased (P>0.05), while intestinal crypt depth was markedly decreased (P<0.001). Lipid droplets in the intestinal villus and mucosal layer in the M8 (8 g/kg) group were more than that in M0 (0 g/kg). Compared with M0 (0 g/kg), the intestinal gcn2 and eif2α were downregulated in M8 (8 g/kg) (P<0.01 and P<0.05, respectively), while occ, cl12, cl15, zo-1, zo-2, hdlbp, ldlrap, npc1l1, cd36, fatp1, fatp2, fatp6, apo, apoa, apob, apoc, apoe, mct1, mct2, mct8, lpl, mttp, moat2, and dgat2 were upregulated markedly in M8 (8 g/kg). Intestinal eif2α expression was positively correlated with gcn2, and intestinal zo-1, cl15, fatp6, ldlrap, mct2, mct8, apo, apob, mct1, apoc, fatp1, mttp, cd36, occ, npc1l1, hdlbp, fatp2, apoe, lpl, and moat2 gene expression was negatively correlated with gcn2. In conclusion, methionine deficiency affected the gastric and intestinal structures, damaged the intestinal barrier, and decreased lipid and fatty acid transport. Besides, gcn2 could be activated when M. albus was fed methionine-deficient feed.


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## Body

## 1. Introduction

With the continuous growth of the aquatic industry, the requirement for fish meals has quickly grown. However, the production of fish meal has continuously decreased, and prices were increased, so the study of replacement of fish meal in aquatic feed has become particularly important [1]. Soybean protein is widely used for replacing fish meal in aquatic feed with high grade of protein [2]. However, methionine is the most deficient amino acid of soybean, which is also an important essential amino acid for aquatic animals ([3]) and must be obtained from feed [4]. Various studies showed that methionine restriction limits protein synthesis, disturbs various metabolism, inhibits growth performance, and damages the healthy state of fish [3, 5, 6], also decreasing intestinal immunomodulatory, digestive, and antioxidant enzymes in rohu (Labeo rohita) [7].In the process of evolution, animals have gradually evolved the ability to adapt to the lack of essential nutrients, such as essential amino acids. A previous study in primary muscular cells of turbot (Scophthalmus maximus L.) demonstrated that methionine restriction reduces cellular lipogenesis while stimulating lipolysis, decreases the content of intracellular lipid, promotes energy expenditure by accelerating progress of tricarboxylic acid cycle and oxidative phosphorylation, and activates the general controlled nonderepressible 2 (gcn2, also encoded by eif2ak4) expression [8]. gcn2 plays a role in vertebrates in response to essential amino acid sensing and metabolism as part of adaptation to nutrient deprivation by regulating its downstream gene expression [9].The gastrointestinal tract is the main position for animals’ digestion and absorption. Gut health has important implications for an animal’s whole healthy statement and utilization of nutrients, because various gastrointestinal functions include digestion and absorption of nutrients by epithelial cells and goblet cells, secretion of mucins and immunoglobulins, and formation of barrier against harmful antigens and pathogens [10]. A previous study showed that different branched-chain amino acids could improve intestinal morphology and cell proliferation, promote intestinal amino acid absorption by regulating intestinal amino acid transporter expression, and increase intestinal protein metabolic efficiency [11]. Methionine is also beneficial for the maintenance of gut morphology and balance of gut bacteria; this system responds to the extensive catabolism of dietary methionine in the gut [12]. A study on nursery pigs showed that supplemented methionine improved small intestinal morphology by increasing villous height and reducing the bacteria fermentation via promoting nutrient digestion and absorption [13]. Dietary methionine could produce glutathione and improve the morphology of the duodenum in nursery pigs [14]. However, there has been little study reporting methionine regulating intestinal health and lipid digestion and absorption, especially for fatty acid transport and absorption in fish.The rice field eel (Monopterus albus, M. albus) is a subtropical freshwater benthic fish and a very economical fish and is in central and southern China, widely raised in cages [15]. M. albus can prey on insects, frog eggs, earthworms, and water earthworms in nature [11] and has a straight tubular gastrointestinal system; we can easily distinguish between stomach and intestine by evident segmentation from anatomy; it also requires better quality and higher levels of protein and also optimum protein/lipid ratio in its diet, as referenced in our previous studies ([16], Hu et al., 2021), which provide an ideal experimental object for our study. Our previous study showed that fish meal was replaced by soybean meal [17] and soy protein concentrate [18] inhibiting the growth performance of M. albus. Our previous study also found that dietary deficiency of methionine in feed decreased the growth performance of M. albus, induced lipid metabolism disorder, and decreased whole-body lipid content accumulation [19]. In this study, we used soy protein concentrate to replace fish meal, designed more serious methionine deficiency diets as we described in a previous study [19], and explored the mechanism of how methionine regulates the intestinal structure and barrier, digestion, lipid transport, and absorption in M. albus.


## 2. Materials and Methods

### 2.1. Experimental Diets

Different levels of methionine (0, 2 g/kg, 4 g/kg, 6 g/kg, 8 g/kg, or 10 g/kg) were supplemented to the basic feed (110 g/kg fish meal; 400 g/kg soy protein concentrate) obeying equal nitrogen and energy based on our previous studies [20]. The composition and nutrition level of the diets are shown in Table 1.Table 1
Composition of the diets and level of nutrition (g/kg).
IngredientsM0M2M4M6M8M10Fish meal110110110110110110Soy protein concentrate400400400400400400Fish oil404040404040DL-Methionine10246810Lysine3.63.63.63.63.63.6Glycine1614121086Glutamate444444Food attractant2111111Wheat meal138.4138.4138.4138.4138.4138.4α-Starch200200200200200200Brewer yeast505050505050Choline chloride555555Ca(H2PO4)2202020202020Vitamin and mineral premix3121212121212Total100010001000100010001000Proximate analysisDry matter (g/kg)922.66925.27928.12928.43923.63924.78Crude protein (g/kg)445.92443.41458.73447.40451.84450.77Crude lipid (g/kg)67.8667.1168.6967.7067.9268.07Crude ash (g/kg)102.60101.90100.60102.60101.90100.60Gross energy (kJ/g)19.1018.8618.7419.1719.2519.101DL-Methionine (BR, 99%): Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). 2Attractants: 40% betaine, 20% DMPT, 20% threonine, 10% glycine, and 10% inosine-5′-diphosphate trisodium salt. 3Vitamin and mineral premix: MGOTer Bio-Tech Co. Ltd (Qingdao, Shandong, China); premix composition (mg/kg diet): KCl, 200 mg; KI (1%), 60 mg; CoCl2·6H2O (1%), 50 mg; CuSO4·5H2O, 30 mg; FeSO4·H2O, 400 mg; ZnSO4·H2O, 400 mg; MnSO4·H2O, 150 mg; Na2SeO3·5H2O (1%), 65 mg; MgSO4·H2O, 2000 mg; zeolite power, 3645.85 mg; VB1, 12 mg; riboflavin, 12 mg; VB6, 8 mg; VB12, 0.05 mg; VK3, 8 mg; inositol, 100 mg; pantothenic acid, 40 mg; niacin acid, 50 mg; folic acid, 5 mg; biotin, 0.8 mg; VA, 25 mg; VCP1, 5 mg; VE, 50 mg; VC, 100 mg; ethoxyquin, 150 mg; wheat meal, 2434.15 mg.Proximate analysis (moisture, crude lipid, crude protein, ash, and gross energy) of experimental feed andM. albus was determined referencing our previous papers (Hu et al., 2021). Amino acids were analyzed by an automatic amino acid analyzer (Agilent-1100, Agilent Technologies Co., Ltd., Santa Clara, CA, USA) referencing the method reported by ([21]); fatty acids were analyzed by GC-MS (Agilent 7890B-5977A, Agilent Technologies Co., Ltd., Santa Clara, CA, USA) referencing the method reported by [22], showed in Tables 2 and 3.Table 2
The contents of amino acids in experimental feed (g/kg).
Amino acidsM0M2M4M6M8M10His☆9.7879.6299.9269.7279.9969.768Ser18.94218.51919.07018.69018.90418.570Arg☆23.41723.85423.42523.19923.53523.118Gly32.73130.51428.36226.27524.13222.012Asp42.24542.15842.10642.71142.53542.631Glu75.48475.67375.21575.74275.91875.681Thr☆15.51415.23015.55615.92515.41215.881Ala19.71819.30119.75919.44719.69719.424Pro20.22719.69720.15320.33020.57520.228Cys1.0841.0291.0881.0911.0841.094Lys☆36.88736.18636.89436.38236.81836.248Tyr9.8029.7599.8529.0409.3979.634Met☆1.8603.7815.9207.7399.60911.525Val☆18.64018.21118.63718.37918.59018.323Ile☆17.47817.13617.61817.63817.89017.465Leu☆29.12529.61229.26729.66629.49329.420Phe☆18.45718.10418.55818.22018.56518.100Trp——————∗Note: ☆ for essential amino acids; Trp not detected.Table 3
The contents of fatty acids in experimental feed (mg/100 g).
Fatty acidsM0M2M4M6M8M10C4:013.2113.7214.4913.5313.1514.16C8:05.075.084.915.055.045.00C12:03.133.644.343.353.354.37C13:011.1310.399.7111.2910.3210.14C14:0181.39183.69182.55182.37183.62182.57C14:12.192.622.812.882.702.83C15:019.9020.2220.5219.9320.2120.51C16:0609.04608.96606.58609.36608.55606.84C16:16.467.596.886.567.586.88C17:012.5813.7413.6512.8013.4213.52C17:16.276.917.336.736.977.38C18:0120.68121.92121.78121.68121.97121.8018:1-T16.1616.0917.8916.1016.0217.86C18:1N9C415.27410.17418.66413.30410.15418.5318:2-T2.743.352.452.733.342.46C18:2N6C17.3516.6318.7118.3416.8618.12C20:011.1310.4510.4910.3010.4010.42C20:125.4427.3727.2723.4327.3427.22C18:3N3235.71235.00236.16235.11236.65235.11C20:210.3510.8810.3110.3610.8510.34C22:05.845.855.955.395.885.91C22:1N9197.83197.62194.40197.33197.65196.49C20:3N332.3731.1734.1932.7433.1334.16C20:4N625.2025.8225.4525.5725.1825.40C24:0248.36249.92237.64248.40249.18237.43C20:5N3101.77100.98101.88101.17101.90101.89C24:121.1921.3622.2921.3921.3223.23C22:6N3575.88571.14571.93575.90571.16570.93


### 2.2. Fish Rearing

M. albus was obtained from Changde, China. We chose a uniform size of M. albus (25.08±0.31 g) randomly distributed into 18 float cages (2.0×1.5×1.5 m), every group including triplicates, 60 fish per cage. For more details, view our previous manuscript [20].


### 2.3. Ethics Statement

This study was supported by the Animal Care Committee of Hunan Agricultural University (Changsha, Hunan Province, China). All experimental fish were anesthetized with eugenol (1 : 12,000) (Shanghai Reagent Corporation, Shanghai, China) before sampling to minimize suffering according to the guidelines established by the National Institutes of Health.


### 2.4. Sample Collection and Analyses

After fasting 24 h, stomach and intestine were obtained from five fish each cage and stored at -80°C until use. Gastric and intestinal digestive enzymes (amylase, lipase and trypsin) were determined by the kit of Nanjing Jiancheng Bioengineering (Nanjing, China).The stomach and intestine from five fish in each cage were taken for histometric evaluation. The method of making slides and observing the intestinal sections stained with H&E referenced our previous manuscript [23]. The intestine was sectioned (8 μm) using a cryostat microtome, stained with Oil red O [24]. The slides were observed by CaseViewer.Total intestinal RNA was obtained from 5 fish in each cage by the Monzol™ reagent (Monad, Shanghai, China). Smart cDNA was synthesized by a SMART cDNA Synthesis kit (Clontech Laboratories, Palo Alto, CA). Primers obtained from Biosune Biotechnology, Inc. (Shanghai, China) are showed in Table4. The operation steps of quantitative real-time PCR (q-PCR) referenced our previous manuscript [25]. The amplification efficiency was between 0.95 and 1.10, which is calculated by the formula E=10∗−1/slope−1; 5-fold serial dilutions of cDNA (triplicate) were used to generate the standard curve. 2-ΔΔCt was used to calculate the relative mRNA expression [26].Table 4
Primer sequence for q-PCR.
GeneForward (5′-3′)Reverse (5′-3′)Accession no.∗Size (bp)gcn21GGAACTCGTCCTGAACTGTGGTGAAGAACTTGCCTATXM_020586241.1298eif2a2CCCCTTCCTTTGTTCGTCGCTGAGGCTTTCTTGTTCCXM_020621840.1121lpl3CGTTGACATCGGAGACCTGACAAAGACCACCTTGGACTGAGXM_020613041.1146moat24TCTCCCTGCCTCTCTTTCATGTCCACTCCATAGTTGCCTXM_020622089.1213dgat25ACTTCCGCTTTCCCTTGATTCCCTGTCTCGTTATGTGXM_020622054.1104mttp6AAGATGCTCCAGGCTTTGTTTGTCAGGACCCTCTAAAATCAGXM_020602163.1172hdlbp7CCACCCCAGACGACAAAGACGGCGAGCAACAAAATAACGAXM_020609988.1165ldlrap8CAGGAAGACAAAAGCAAGAAGGCGAGTGGGGTTACTATGAGGCXM_020617284.1194vldlr9ACATCCGTCGTTTGGGTCTAGTGGTAGTGTCCCCTCGTTTXM_020601062.1169npc1l110TTGGAGTCCCAGTTTATTTTACACTTGCGTCCACATTXM_020590431.1297mct111TCCTATGCCTTCCCTAAATAAGTTGAATGCCAGTCCCXM_020586598.1287mct212TGGGCTTGTCACCATTATCTCCTCGTCCAGTTTCTTXM_020589687.1181mct413GAGGAGCAGTGGTGGATGGGGAAGGCGTAGGAGAAAXM_020608921.1112mct714GTTGTCATTGGCACCCTTACCTGAGTCCTCCGAACCXM_020608232.1210mct815CAGCAGGACCTTCCAAATAAAGTAGCCCAGGACAGCXM_020592011.1271mct1216GTTGGCGTATGGGATTGCTTTGGCGAGATTTGGATGTXM_020616669.1223cd3617TTGAAAGGGATTGAGGTGTCTCGCAAGGATGGACTAXM_020616796.1212fatp118GCGAGCCAGGTATGTTAGCAGCAAGGCACTGAGGACXM_020587461.1263fatp219CTTTGATTACAGCCTTGCCTTTCCGTTGTCCTTTCTXM_020602138.1100fatp620CAGTAGGACTTTGGGCATTTGTCGCACTTTGTGAACTTTATCXM_020618747.1267fatp721ACTGTAATCATCAGCCAAGAGGTTTCGTCAAACTCCTCXM_020588511.1105apo22GGGCTGCTCTGGATGTCTCCCGCAAAGCACTAATCTHQ603782.1147apoa23CAAGAAGGTCCAGGTTGATTAGTAAGGGATTGGTAGAGGXM_020590134.1147apob24TGCCAATAACTATCCGCTACTCTTCCTGACATCATCCCXM_020615697.1247apoc25GCTGCTGGTCGTTACTGTAGTCCCTAATGGTTTCTATGXM_020590135.1176apoe26CGCTGCGTGGAAGGAAACCTGCCAGAGCAAGGATGAGAXM_020590131.1220apof27AGGTGGTAAGCCTGATAGACCAACCCTCATAGTGTCCXM_020592847.1185apoo28GCTCAGGTTCGGTTTGTTGGTGGCAACTCTGGGTATXM_020595548.1207occ29TGTCGGGGAGTGGGTAAATCCAGGCAAATAAAGAGGCTXM_020616177.1130zo-130GGCATCATCCCCAACAAAGCGAAGACCACGGAACCTXM_020621576.1111zo-231AGCCGAGGTCGCACTTTAGCTTTGCTTCTGTGGTTGATXM_020615114.1246cl-1232TCACCTTCAATCGCAACGATGTCTGGCTCAGGCTTATCTXM_020607277.1250cl-1533CTCGCTGCTTGCTTTGACTTTGAAGGCGTACCAGGACAXM_020611334.1225rpL1734CGAGAACCCGACTAAATCAGTTGTAGCGACGGAAAGGXM_020587712.11691gcn2: general control nonderepressible; 2eif2a: eukaryotic translation initiation factor 2; 3lpl: lipoprotein lipase; 4moat2: monoacylglycerol O-acyltransferase 2; 5dgat2: diacylglycerol acyltransferase 2; 6mttp: microsomal triglyceride transfer protein; 7hdlbp: high-density lipoprotein binding protein; 8ldlrap: low-density lipoprotein receptor adapter protein; 9vldlr: very low-density lipoprotein receptor; 10npc1l1: NPC1 like intracellular cholesterol transporter 1; 11mct1: monocarboxylate transporter 1-like; 12mct2: monocarboxylate transporter 2-like; 13mct4: monocarboxylate transporter 4-like; 14mct7: monocarboxylate transporter 7-like; 15mct8: monocarboxylate transporter 8-like; 16mct12: monocarboxylate transporter 12-B-like; 17cd36: CD36 molecule; 18fatp1: fatty acid transport protein 1; 19fatp2: fatty acid binding protein 2; 20fatp6: fatty acid transport protein 6; 21fatp7: fatty acid binding protein 7; 22apo: apolipoprotein; 23apoa: apolipoprotein A; 24apob: apolipoprotein B; 25apoc: apolipoprotein C; 26apoe: apolipoprotein E. 27apof: apolipoprotein F; 28apoo: apolipoprotein O; 29occ: occludin-like; 30zo-1: tight junction protein ZO-1-like; 31zo-2: tight junction protein ZO-2-like; 32cl-12: claudin 12; 33cl-15: claudin 15; 34rpL17: ribosomal protein L17; it is reference gene. ∗NCBI Reference Sequence.


### 2.5. Statistical Analysis

Data were analyzed by one-way analysis of variance (ANOVA), and significant differences among all groups were assessed by Duncan’s multiple-range test; the data of two groups (M0 and M8) was calculated by an independentT-test; ANOVA and independent T-test were performed by SPSS 22 software. The results were presented as the means±SEM (standard error of the mean), and differences were considered significant at P<0.05.


## 2.1. Experimental Diets

Different levels of methionine (0, 2 g/kg, 4 g/kg, 6 g/kg, 8 g/kg, or 10 g/kg) were supplemented to the basic feed (110 g/kg fish meal; 400 g/kg soy protein concentrate) obeying equal nitrogen and energy based on our previous studies [20]. The composition and nutrition level of the diets are shown in Table 1.Table 1
Composition of the diets and level of nutrition (g/kg).
IngredientsM0M2M4M6M8M10Fish meal110110110110110110Soy protein concentrate400400400400400400Fish oil404040404040DL-Methionine10246810Lysine3.63.63.63.63.63.6Glycine1614121086Glutamate444444Food attractant2111111Wheat meal138.4138.4138.4138.4138.4138.4α-Starch200200200200200200Brewer yeast505050505050Choline chloride555555Ca(H2PO4)2202020202020Vitamin and mineral premix3121212121212Total100010001000100010001000Proximate analysisDry matter (g/kg)922.66925.27928.12928.43923.63924.78Crude protein (g/kg)445.92443.41458.73447.40451.84450.77Crude lipid (g/kg)67.8667.1168.6967.7067.9268.07Crude ash (g/kg)102.60101.90100.60102.60101.90100.60Gross energy (kJ/g)19.1018.8618.7419.1719.2519.101DL-Methionine (BR, 99%): Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). 2Attractants: 40% betaine, 20% DMPT, 20% threonine, 10% glycine, and 10% inosine-5′-diphosphate trisodium salt. 3Vitamin and mineral premix: MGOTer Bio-Tech Co. Ltd (Qingdao, Shandong, China); premix composition (mg/kg diet): KCl, 200 mg; KI (1%), 60 mg; CoCl2·6H2O (1%), 50 mg; CuSO4·5H2O, 30 mg; FeSO4·H2O, 400 mg; ZnSO4·H2O, 400 mg; MnSO4·H2O, 150 mg; Na2SeO3·5H2O (1%), 65 mg; MgSO4·H2O, 2000 mg; zeolite power, 3645.85 mg; VB1, 12 mg; riboflavin, 12 mg; VB6, 8 mg; VB12, 0.05 mg; VK3, 8 mg; inositol, 100 mg; pantothenic acid, 40 mg; niacin acid, 50 mg; folic acid, 5 mg; biotin, 0.8 mg; VA, 25 mg; VCP1, 5 mg; VE, 50 mg; VC, 100 mg; ethoxyquin, 150 mg; wheat meal, 2434.15 mg.Proximate analysis (moisture, crude lipid, crude protein, ash, and gross energy) of experimental feed andM. albus was determined referencing our previous papers (Hu et al., 2021). Amino acids were analyzed by an automatic amino acid analyzer (Agilent-1100, Agilent Technologies Co., Ltd., Santa Clara, CA, USA) referencing the method reported by ([21]); fatty acids were analyzed by GC-MS (Agilent 7890B-5977A, Agilent Technologies Co., Ltd., Santa Clara, CA, USA) referencing the method reported by [22], showed in Tables 2 and 3.Table 2
The contents of amino acids in experimental feed (g/kg).
Amino acidsM0M2M4M6M8M10His☆9.7879.6299.9269.7279.9969.768Ser18.94218.51919.07018.69018.90418.570Arg☆23.41723.85423.42523.19923.53523.118Gly32.73130.51428.36226.27524.13222.012Asp42.24542.15842.10642.71142.53542.631Glu75.48475.67375.21575.74275.91875.681Thr☆15.51415.23015.55615.92515.41215.881Ala19.71819.30119.75919.44719.69719.424Pro20.22719.69720.15320.33020.57520.228Cys1.0841.0291.0881.0911.0841.094Lys☆36.88736.18636.89436.38236.81836.248Tyr9.8029.7599.8529.0409.3979.634Met☆1.8603.7815.9207.7399.60911.525Val☆18.64018.21118.63718.37918.59018.323Ile☆17.47817.13617.61817.63817.89017.465Leu☆29.12529.61229.26729.66629.49329.420Phe☆18.45718.10418.55818.22018.56518.100Trp——————∗Note: ☆ for essential amino acids; Trp not detected.Table 3
The contents of fatty acids in experimental feed (mg/100 g).
Fatty acidsM0M2M4M6M8M10C4:013.2113.7214.4913.5313.1514.16C8:05.075.084.915.055.045.00C12:03.133.644.343.353.354.37C13:011.1310.399.7111.2910.3210.14C14:0181.39183.69182.55182.37183.62182.57C14:12.192.622.812.882.702.83C15:019.9020.2220.5219.9320.2120.51C16:0609.04608.96606.58609.36608.55606.84C16:16.467.596.886.567.586.88C17:012.5813.7413.6512.8013.4213.52C17:16.276.917.336.736.977.38C18:0120.68121.92121.78121.68121.97121.8018:1-T16.1616.0917.8916.1016.0217.86C18:1N9C415.27410.17418.66413.30410.15418.5318:2-T2.743.352.452.733.342.46C18:2N6C17.3516.6318.7118.3416.8618.12C20:011.1310.4510.4910.3010.4010.42C20:125.4427.3727.2723.4327.3427.22C18:3N3235.71235.00236.16235.11236.65235.11C20:210.3510.8810.3110.3610.8510.34C22:05.845.855.955.395.885.91C22:1N9197.83197.62194.40197.33197.65196.49C20:3N332.3731.1734.1932.7433.1334.16C20:4N625.2025.8225.4525.5725.1825.40C24:0248.36249.92237.64248.40249.18237.43C20:5N3101.77100.98101.88101.17101.90101.89C24:121.1921.3622.2921.3921.3223.23C22:6N3575.88571.14571.93575.90571.16570.93


## 2.2. Fish Rearing

M. albus was obtained from Changde, China. We chose a uniform size of M. albus (25.08±0.31 g) randomly distributed into 18 float cages (2.0×1.5×1.5 m), every group including triplicates, 60 fish per cage. For more details, view our previous manuscript [20].


## 2.3. Ethics Statement

This study was supported by the Animal Care Committee of Hunan Agricultural University (Changsha, Hunan Province, China). All experimental fish were anesthetized with eugenol (1 : 12,000) (Shanghai Reagent Corporation, Shanghai, China) before sampling to minimize suffering according to the guidelines established by the National Institutes of Health.


## 2.4. Sample Collection and Analyses

After fasting 24 h, stomach and intestine were obtained from five fish each cage and stored at -80°C until use. Gastric and intestinal digestive enzymes (amylase, lipase and trypsin) were determined by the kit of Nanjing Jiancheng Bioengineering (Nanjing, China).The stomach and intestine from five fish in each cage were taken for histometric evaluation. The method of making slides and observing the intestinal sections stained with H&E referenced our previous manuscript [23]. The intestine was sectioned (8 μm) using a cryostat microtome, stained with Oil red O [24]. The slides were observed by CaseViewer.Total intestinal RNA was obtained from 5 fish in each cage by the Monzol™ reagent (Monad, Shanghai, China). Smart cDNA was synthesized by a SMART cDNA Synthesis kit (Clontech Laboratories, Palo Alto, CA). Primers obtained from Biosune Biotechnology, Inc. (Shanghai, China) are showed in Table4. The operation steps of quantitative real-time PCR (q-PCR) referenced our previous manuscript [25]. The amplification efficiency was between 0.95 and 1.10, which is calculated by the formula E=10∗−1/slope−1; 5-fold serial dilutions of cDNA (triplicate) were used to generate the standard curve. 2-ΔΔCt was used to calculate the relative mRNA expression [26].Table 4
Primer sequence for q-PCR.
GeneForward (5′-3′)Reverse (5′-3′)Accession no.∗Size (bp)gcn21GGAACTCGTCCTGAACTGTGGTGAAGAACTTGCCTATXM_020586241.1298eif2a2CCCCTTCCTTTGTTCGTCGCTGAGGCTTTCTTGTTCCXM_020621840.1121lpl3CGTTGACATCGGAGACCTGACAAAGACCACCTTGGACTGAGXM_020613041.1146moat24TCTCCCTGCCTCTCTTTCATGTCCACTCCATAGTTGCCTXM_020622089.1213dgat25ACTTCCGCTTTCCCTTGATTCCCTGTCTCGTTATGTGXM_020622054.1104mttp6AAGATGCTCCAGGCTTTGTTTGTCAGGACCCTCTAAAATCAGXM_020602163.1172hdlbp7CCACCCCAGACGACAAAGACGGCGAGCAACAAAATAACGAXM_020609988.1165ldlrap8CAGGAAGACAAAAGCAAGAAGGCGAGTGGGGTTACTATGAGGCXM_020617284.1194vldlr9ACATCCGTCGTTTGGGTCTAGTGGTAGTGTCCCCTCGTTTXM_020601062.1169npc1l110TTGGAGTCCCAGTTTATTTTACACTTGCGTCCACATTXM_020590431.1297mct111TCCTATGCCTTCCCTAAATAAGTTGAATGCCAGTCCCXM_020586598.1287mct212TGGGCTTGTCACCATTATCTCCTCGTCCAGTTTCTTXM_020589687.1181mct413GAGGAGCAGTGGTGGATGGGGAAGGCGTAGGAGAAAXM_020608921.1112mct714GTTGTCATTGGCACCCTTACCTGAGTCCTCCGAACCXM_020608232.1210mct815CAGCAGGACCTTCCAAATAAAGTAGCCCAGGACAGCXM_020592011.1271mct1216GTTGGCGTATGGGATTGCTTTGGCGAGATTTGGATGTXM_020616669.1223cd3617TTGAAAGGGATTGAGGTGTCTCGCAAGGATGGACTAXM_020616796.1212fatp118GCGAGCCAGGTATGTTAGCAGCAAGGCACTGAGGACXM_020587461.1263fatp219CTTTGATTACAGCCTTGCCTTTCCGTTGTCCTTTCTXM_020602138.1100fatp620CAGTAGGACTTTGGGCATTTGTCGCACTTTGTGAACTTTATCXM_020618747.1267fatp721ACTGTAATCATCAGCCAAGAGGTTTCGTCAAACTCCTCXM_020588511.1105apo22GGGCTGCTCTGGATGTCTCCCGCAAAGCACTAATCTHQ603782.1147apoa23CAAGAAGGTCCAGGTTGATTAGTAAGGGATTGGTAGAGGXM_020590134.1147apob24TGCCAATAACTATCCGCTACTCTTCCTGACATCATCCCXM_020615697.1247apoc25GCTGCTGGTCGTTACTGTAGTCCCTAATGGTTTCTATGXM_020590135.1176apoe26CGCTGCGTGGAAGGAAACCTGCCAGAGCAAGGATGAGAXM_020590131.1220apof27AGGTGGTAAGCCTGATAGACCAACCCTCATAGTGTCCXM_020592847.1185apoo28GCTCAGGTTCGGTTTGTTGGTGGCAACTCTGGGTATXM_020595548.1207occ29TGTCGGGGAGTGGGTAAATCCAGGCAAATAAAGAGGCTXM_020616177.1130zo-130GGCATCATCCCCAACAAAGCGAAGACCACGGAACCTXM_020621576.1111zo-231AGCCGAGGTCGCACTTTAGCTTTGCTTCTGTGGTTGATXM_020615114.1246cl-1232TCACCTTCAATCGCAACGATGTCTGGCTCAGGCTTATCTXM_020607277.1250cl-1533CTCGCTGCTTGCTTTGACTTTGAAGGCGTACCAGGACAXM_020611334.1225rpL1734CGAGAACCCGACTAAATCAGTTGTAGCGACGGAAAGGXM_020587712.11691gcn2: general control nonderepressible; 2eif2a: eukaryotic translation initiation factor 2; 3lpl: lipoprotein lipase; 4moat2: monoacylglycerol O-acyltransferase 2; 5dgat2: diacylglycerol acyltransferase 2; 6mttp: microsomal triglyceride transfer protein; 7hdlbp: high-density lipoprotein binding protein; 8ldlrap: low-density lipoprotein receptor adapter protein; 9vldlr: very low-density lipoprotein receptor; 10npc1l1: NPC1 like intracellular cholesterol transporter 1; 11mct1: monocarboxylate transporter 1-like; 12mct2: monocarboxylate transporter 2-like; 13mct4: monocarboxylate transporter 4-like; 14mct7: monocarboxylate transporter 7-like; 15mct8: monocarboxylate transporter 8-like; 16mct12: monocarboxylate transporter 12-B-like; 17cd36: CD36 molecule; 18fatp1: fatty acid transport protein 1; 19fatp2: fatty acid binding protein 2; 20fatp6: fatty acid transport protein 6; 21fatp7: fatty acid binding protein 7; 22apo: apolipoprotein; 23apoa: apolipoprotein A; 24apob: apolipoprotein B; 25apoc: apolipoprotein C; 26apoe: apolipoprotein E. 27apof: apolipoprotein F; 28apoo: apolipoprotein O; 29occ: occludin-like; 30zo-1: tight junction protein ZO-1-like; 31zo-2: tight junction protein ZO-2-like; 32cl-12: claudin 12; 33cl-15: claudin 15; 34rpL17: ribosomal protein L17; it is reference gene. ∗NCBI Reference Sequence.


## 2.5. Statistical Analysis

Data were analyzed by one-way analysis of variance (ANOVA), and significant differences among all groups were assessed by Duncan’s multiple-range test; the data of two groups (M0 and M8) was calculated by an independentT-test; ANOVA and independent T-test were performed by SPSS 22 software. The results were presented as the means±SEM (standard error of the mean), and differences were considered significant at P<0.05.


## 3. Result

### 3.1. Gastric and Intestinal Digestive Enzymes

Compared with M0 (0 g/kg), gastric amylase, lipase, and trypsin activities were remarkably increased as dietary methionine (P<0.05); intestinal amylase was remarkably increased as added 8 g/kg methionine (P<0.05), intestinal lipase was remarkably increased as supplemented methionine higher than 2 g/kg (P<0.05), and intestinal trypsin was remarkably increased as dietary methionine (P<0.05) (Table 5).Table 5
Effects of different levels of methionine on gastric and intestinal digestive enzymes ofM. albus after 8 weeks (U/g protein).
ItemM0M2M4M6M8M10P valueStomachAmylase231.94±5.14a257.95±4.87b289.7±3.72c299.84±3.78c358.05±4.46d357.61±4.07d<0.001Lipase427.83±11.73a474.53±5.54b485.74±4.73b513.76±11.36c566.08±5.61d586.63±3.74d<0.001Trypsin2156.23±22.47a2244.78±23.86b2438.06±43.50c3081.08±20.33d3423.03±14.14e3467.2±43.26e<0.001IntestineAmylase270.7±3.84a275.38±3.55a278.51±4.37a281.63±3.99ab294.12±3.17c291±3.55bc0.001Lipase335.53±3.79a347.1±3.99a363.93±9.45b411.26±3.79c429.15±2.30d439.67±2.66d<0.001Trypsin2161.05±32.27a2249.94±36.82b2273.27±14.48b2445.51±18.24c2457.73±15.94c2472.73±14.26c<0.001Values showed asmeans±SEMn=3. Values in the same row with the same superscript or absence of superscripts are not significantly different (P>0.05).


### 3.2. Gastric and Intestinal Sections Stained with H&E

The results of gastric and intestinal sections stained with H&E are presented in Figures1 and 2. Compared with M0 (0 g/kg), gastric fovea increased remarkably in M8 (8 g/kg) (P<0.001); gastric epithelium is neater in M8 than that in M0 (Table 6). Compared with M0, intestinal villus height and intestinal muscular thickness remarkably increased in M8 (P<0.001), and amounts of goblet cells per root in M8 increased (P>0.05), while intestinal crypt depth markedly decreased (P<0.001) (Table 7). In addition, lipid droplets in intestinal villus and mucosal layer in the M8 (8 g/kg) group were more than that in M0 (0 g/kg) (Figure 3).Figure 1
Effects of deficient and optimum methionine diet on the gastric H&E stain ofM. albus after 8 weeks. (1) and (2) were expressed as gastric fovea and gastric muscular thickness, respectively.Figure 2
Effects of deficient and optimum methionine diet on the intestinal H&E stain ofM. albus after 8 weeks. (1), (2), (3), and (4) were expressed as intestinal villus height, crypt depth, muscular thickness, and goblet cell, respectively.Table 6
Effects of deficient and optimum methionine diet on the gastric H&E stain (×100 and ×400) ofM. albus after 8 weeks (n=3).
ItemM0M8P valueGF1440.11±27.08648.31±24.30<0.001GMT294.48±0.9897.31±1.770.193Values are presented asmeans±SEMn=3. Values were considered not significantly different at P>0.05. 1GF: gastric fovea (μm); 2GMT: gastric muscular thickness (μm).Table 7
Effects of deficient and optimum methionine diet on the intestinal sections stained with H&E (×100 and ×400) ofM. albus after 8 weeks (n=3).
ItemM0M8P valueIVH1380.71±12.14693.54±21.81<0.001CD247.12±1.6332.82±0.91<0.001IMT353.11±3.4094.53±2.00<0.001AIGC427±333±20.084Values showed asmeans±SEMn=3. Values were considered not significantly different at P>0.05. 1IVH: intestinal villus height (μm); 2CD: crypt depth (μm); 3IMT: intestinal muscular thickness (μm); 4AIGC: amounts of intestinal goblet cells per root.Figure 3
Effects of deficient and optimum methionine diet on the intestinal Oil red O stain (×100) ofM. albus after 8 weeks.


### 3.3. Intestinal Regulatory mRNA Expression

Compared with M0 (0 g/kg), the intestinalgcn2 and eif2α are downregulated in M8 (8 g/kg) (P<0.01 and P<0.05, respectively), while occ, cl12, cl15, zo-1, zo-2, hdlbp, ldlrap, npc1l1, cd36, fatp1, fatp2, fatp6, apo, apoa, apob, apoc, apoe, mct1, mct2, mct8, lpl, mttp, moat2, and dgat2 were upregulated markedly in M8 (8 g/kg) (Figure 4).Figure 4
Effects of dietary methionine on intestinal mRNA expression ofM. albus after 8 weeks (n=3). Single, double, or triple numbers of asterisks were significantly different at P<0.05, P<0.01, and P<0.001, respectively.
(a)(b)(c)(d)(e)(f)(g)


### 3.4. Intestinal Regulatory mRNA Expression

We observed that intestinaleif2α expression was positively correlated with gcn2, and intestinal zo-1, cl15, fatp6, ldlrap, mct2, mct8, apo, apob, mct1, apoc, fatp1, mttp, cd36, occ, npc1l1, hdlbp, fatp2, apoe, lpl, and moat2 gene expression was negatively correlated with gcn2 (P<0.05,P<0.05,P<0.05,P<0.05,P<0.01,P<0.01,P<0.05,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.05,P<0.05,P<0.05,P<0.01,P<0.05,P<0.05,andP<0.05) (Figure 5).Figure 5
Correlative analysis of intestinal regulatory mRNA expression used by R Programming Language. Single, double, or triple numbers of asterisks were significantly different atP<0.05,P<0.01,andP<0.001, respectively.


## 3.1. Gastric and Intestinal Digestive Enzymes

Compared with M0 (0 g/kg), gastric amylase, lipase, and trypsin activities were remarkably increased as dietary methionine (P<0.05); intestinal amylase was remarkably increased as added 8 g/kg methionine (P<0.05), intestinal lipase was remarkably increased as supplemented methionine higher than 2 g/kg (P<0.05), and intestinal trypsin was remarkably increased as dietary methionine (P<0.05) (Table 5).Table 5
Effects of different levels of methionine on gastric and intestinal digestive enzymes ofM. albus after 8 weeks (U/g protein).
ItemM0M2M4M6M8M10P valueStomachAmylase231.94±5.14a257.95±4.87b289.7±3.72c299.84±3.78c358.05±4.46d357.61±4.07d<0.001Lipase427.83±11.73a474.53±5.54b485.74±4.73b513.76±11.36c566.08±5.61d586.63±3.74d<0.001Trypsin2156.23±22.47a2244.78±23.86b2438.06±43.50c3081.08±20.33d3423.03±14.14e3467.2±43.26e<0.001IntestineAmylase270.7±3.84a275.38±3.55a278.51±4.37a281.63±3.99ab294.12±3.17c291±3.55bc0.001Lipase335.53±3.79a347.1±3.99a363.93±9.45b411.26±3.79c429.15±2.30d439.67±2.66d<0.001Trypsin2161.05±32.27a2249.94±36.82b2273.27±14.48b2445.51±18.24c2457.73±15.94c2472.73±14.26c<0.001Values showed asmeans±SEMn=3. Values in the same row with the same superscript or absence of superscripts are not significantly different (P>0.05).


## 3.2. Gastric and Intestinal Sections Stained with H&E

The results of gastric and intestinal sections stained with H&E are presented in Figures1 and 2. Compared with M0 (0 g/kg), gastric fovea increased remarkably in M8 (8 g/kg) (P<0.001); gastric epithelium is neater in M8 than that in M0 (Table 6). Compared with M0, intestinal villus height and intestinal muscular thickness remarkably increased in M8 (P<0.001), and amounts of goblet cells per root in M8 increased (P>0.05), while intestinal crypt depth markedly decreased (P<0.001) (Table 7). In addition, lipid droplets in intestinal villus and mucosal layer in the M8 (8 g/kg) group were more than that in M0 (0 g/kg) (Figure 3).Figure 1
Effects of deficient and optimum methionine diet on the gastric H&E stain ofM. albus after 8 weeks. (1) and (2) were expressed as gastric fovea and gastric muscular thickness, respectively.Figure 2
Effects of deficient and optimum methionine diet on the intestinal H&E stain ofM. albus after 8 weeks. (1), (2), (3), and (4) were expressed as intestinal villus height, crypt depth, muscular thickness, and goblet cell, respectively.Table 6
Effects of deficient and optimum methionine diet on the gastric H&E stain (×100 and ×400) ofM. albus after 8 weeks (n=3).
ItemM0M8P valueGF1440.11±27.08648.31±24.30<0.001GMT294.48±0.9897.31±1.770.193Values are presented asmeans±SEMn=3. Values were considered not significantly different at P>0.05. 1GF: gastric fovea (μm); 2GMT: gastric muscular thickness (μm).Table 7
Effects of deficient and optimum methionine diet on the intestinal sections stained with H&E (×100 and ×400) ofM. albus after 8 weeks (n=3).
ItemM0M8P valueIVH1380.71±12.14693.54±21.81<0.001CD247.12±1.6332.82±0.91<0.001IMT353.11±3.4094.53±2.00<0.001AIGC427±333±20.084Values showed asmeans±SEMn=3. Values were considered not significantly different at P>0.05. 1IVH: intestinal villus height (μm); 2CD: crypt depth (μm); 3IMT: intestinal muscular thickness (μm); 4AIGC: amounts of intestinal goblet cells per root.Figure 3
Effects of deficient and optimum methionine diet on the intestinal Oil red O stain (×100) ofM. albus after 8 weeks.


## 3.3. Intestinal Regulatory mRNA Expression

Compared with M0 (0 g/kg), the intestinalgcn2 and eif2α are downregulated in M8 (8 g/kg) (P<0.01 and P<0.05, respectively), while occ, cl12, cl15, zo-1, zo-2, hdlbp, ldlrap, npc1l1, cd36, fatp1, fatp2, fatp6, apo, apoa, apob, apoc, apoe, mct1, mct2, mct8, lpl, mttp, moat2, and dgat2 were upregulated markedly in M8 (8 g/kg) (Figure 4).Figure 4
Effects of dietary methionine on intestinal mRNA expression ofM. albus after 8 weeks (n=3). Single, double, or triple numbers of asterisks were significantly different at P<0.05, P<0.01, and P<0.001, respectively.
(a)(b)(c)(d)(e)(f)(g)


## 3.4. Intestinal Regulatory mRNA Expression

We observed that intestinaleif2α expression was positively correlated with gcn2, and intestinal zo-1, cl15, fatp6, ldlrap, mct2, mct8, apo, apob, mct1, apoc, fatp1, mttp, cd36, occ, npc1l1, hdlbp, fatp2, apoe, lpl, and moat2 gene expression was negatively correlated with gcn2 (P<0.05,P<0.05,P<0.05,P<0.05,P<0.01,P<0.01,P<0.05,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.05,P<0.05,P<0.05,P<0.01,P<0.05,P<0.05,andP<0.05) (Figure 5).Figure 5
Correlative analysis of intestinal regulatory mRNA expression used by R Programming Language. Single, double, or triple numbers of asterisks were significantly different atP<0.05,P<0.01,andP<0.001, respectively.


## 4. Discussions

Methionine is the most deficient essential amino acid of most plant proteins, especially for soybean protein [27]. Our previous study showed that dietary methionine restriction not only restricted muscle fiber growth, muscular development, and differentiation of M. albus and inhibited growth performance of M. albus [20] but also induced lipid metabolism disorder and decreased lipid content of M. albus (Hu et al., 2021). Our previous study showed that crude lipid and crude protein of M. albus were significantly promoted as supplemented with 8 g/kg methionine. In this study, compared with M0 (0 g/kg), gastric amylase, lipase, and trypsin were remarkably increased as dietary methionine; intestinal amylase, lipase, and trypsin were remarkably increased as dietary high than the level of 8 g/kg (M8) methionine. Our finding was similar to the study in grass carp (Ctenopharyngodon idella) [28]. We hold that methionine deficiency decreased gastric-intestinal main digestive enzymes (amylase, lipase, and trypsin) of M. albus and mainly affected the stomach.Based on the photos of intestinal H&E staining, the intestinal lumen in M8 (8 g/kg) was bigger than that in M0 (0 g/kg); the reason was that the fish were smaller in M0 than that in M8 because methionine restriction inhibited the growth performance ofM. Albus as we early reported [20]. Here, compared with M0 (0 g/kg), gastric fovea increased remarkably in M8 (8 g/kg); gastric epithelium is neater in M8 than that in M0, which meant that the capacity of the gastric digestive system became weak; this phenomenon explained that amylase, lipase, and trypsin were remarkably decreased as methionine restriction. In addition, in this paper, compared with M0, we also observed that intestinal villus height and muscular thickness were remarkably increased in M8, and amounts of goblet cells per root in M8 were increased, while intestinal crypt depth remarkably decreased; besides, lipid droplet in intestinal villus and mucosal layer in the M8 (8 g/kg) group were more than that in M0 (0 g/kg) in this study, which meant that the function of intestinal absorption was declined and the intestinal barrier was damaged [29], also including lipid. Our previous study showed a similar result as dietary soy isoflavone and soy saponin damage the intestinal barrier and decrease intestinal function [23].To further explain the reasons how methionine restriction influences gastrointestinal lipid digestion and absorption ofM. albus, the M0 (0 g/kg) and M8 (8 g/kg) groups were selected to explore the molecular mechanism. gcn2 and eif2a are a response to essential amino acid deprivation and regulate its downstream lipid metabolism relative genes [30]. In this study, compared with M0 (0 g/kg), the intestinal gcn2 and eif2α are downregulated in M8 (8 g/kg); this meant that amino acid deficiency can be sensed by M. albus. Intestinal tight junction protein includes occludens, claudin, and zonula, could form the epithelial barrier and prevent infiltration, and is indispensable in protecting barrier integrity and function [31]. We observed that occ, cl12, cl15, zo-1, and zo-2 genes are upregulated markedly in M8 (8 g/kg), which explained that methionine deficiency affected gastric and intestinal structures and damaged the intestinal barrier. As we know, lipid can be digested into fatty acids and alcohols in the intestine; then, fatty acids and alcohols were absorbed, and fatty acids and alcohols assemble into lipid; eventually, this lipid transports into the whole body by blood circulation. Microsomal triglyceride transfer protein (mttp) facilitates the transport of fat by assisting in the assembly and secretion of triglyceride-rich lipoproteins [32]. Lipoprotein lipase (lpl) is involved in lipolysis [33], while mogat2 and dgat2 participate in lipogenesis [34, 35]. In this study, intestinal mttp, lpl, moat2, and dgat2 were upregulated markedly in M8 (8 g/kg), which indicated that lipid metabolism is more active as a dietary appropriate level of methionine. This phenomenon indirectly explained why supplementation with methionine enhanced gastric and intestinal digestive enzymes and promoted the ability of digestion in our study. High-density lipoprotein-binding protein (hdlbp) regulates the endocrine of both lipids and cholesterol ([36]); the low-density lipoprotein receptor adapter protein (ldlra) pathway has emerged as a target to reduce circulating cholesterol [37]. NPC1 like intracellular cholesterol transporter 1 (npc1l1) is mainly charged in cholesterol absorption [38]. CD36 molecule (cd36) plays a role in fatty acid absorption and transport [39, 40]. Apolipoprotein can bind and transport lipoproteins [41]. Monocarboxylate transporter mainly transports short-chain monocarboxylates, including lactate, pyruvate, and ketone bodies [42]. In this study, intestinal hdlbp, ldlrap, npc1l1, cd36, fatp1, fatp2, fatp6, apo, apoa, apob, apoc, apoe, mct1, mct2, and mct8 were upregulated in M8 (8 g/kg) than that in M0 (0 g/kg). We inferred that methionine deficiency suppressed intestinal mucosal growth and inhibits intestinal epithelial cell proliferation, damaged the intestinal barrier [29, 43], and then declined intestinal lipid and fatty acid transport.We observed that intestinaleif2α expression was positively correlated with gcn2, and intestinal zo-1, cl15, fatp6, ldlrap, mct2, mct8, apo, apob, mct1, apoc, fatp1, mttp, cd36, occ, npc1l1, hdlbp, fatp2, apoe, lpl, and moat2 gene expression was negatively correlated with gcn2. We inferred that M. albus could sense methionine deficiency by gcn2 and regulated the lipid and fatty acid transport.


## 5. Conclusion

Methionine deficiency mainly affected gastric and intestinal structures, damaged the intestinal barrier, and decreased the lipid and fatty acid transport ofM. albus. Besides, gcn2 could be activated when M. albus was fed methionine-deficient feed.



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*Source: 2293484-2022-02-15.xml*