Comparative study of leucine on digestive enzyme secretion, ISR and mTOR pathway in cattle and porcine pancreas in vitro

Figures & data

Table 1. Ingredients and chemical composition of the cattle and pigs (air-dry basis).

Ingredients	Cattle	Pigs	Chemical composition,%	Cattle	Pigs
Corn	8.00	34.96	DM	59.71	89.88
Tablet corn	14.73		CP	15.83	12.09
Soybean meal	11.25	3.94	Ash	6.97	5.78
DDGS	5.00		NDF	39.68	38.92
Cottonseed meal	4.00	3.94	ADF	19.19	21.18
Beet pulp	5.00
Alfalfa hay	22.00
Corn silage	18.00	35.41
Extruded soybean	2.63
Wheat bran		4.76
Distillers grain
Premix^a	2.06	0.5.
Sodium hydrogen carbonate	0.62	0.53
sodium chloride	0.29	0.53
Calcium hydrogen phosphate	0.21	0.79

^a Premix provides the following (unit/kg of diet) of cattle: VA, 6030 IU; VD₃, 1935 IU; VE, 18 IU; VB₅, 17.91 IU; Biotin, 0.07 IU; Cu, 5.2 mg; Fe, 24.6 mg; Mn, 23.7 mg; Co, 0.16 mg; Se, 0.34 mg. Premix provided the following (unit/kg of diet) of pigs: VA, 6400 IU; VD₃, 2000 IU; VE, 30 IU; VK₃, 3 mg; VB₁₂, 0.03 mg; VB₂, 5.5 mg; nicotinic acid, 27 mg; pantothenic acid, 14 mg; folic acid, 0.68 mg, VB₁, 0.8 mg; VB₆, 0.8 mg; biotin, 0.05 mg; Zn, 90 mg; Mn, 30 mg; Fe, 100 mg; Cu, 20 mg; I, 0.40 mg; Se, 0.30 mg.

Table 2. The PCR primers used to amplify the genes encoding PKB, 4E-BP1, S6K1, eIF-2α, ATF4 and β-actin in pig.

Target genes	Reference sequence	Primer sequence (5'→3')	Product size (bp)	Annealing temperature(°C)
PKB	NM_001159776.1	GGTATTTCGACGAGGAGTTCAC GAGTAGGAGAACTGGGGGAAGT	118	60
4E-BP1	NM_001244225.1	GGGGACTACAGCACCACTCC GCTTGTCGTCTGGGCTACTG	223	59
S6K1	XM_003131671.5	GGGGATCATCTACAGAGACCTG CCACAAAATGTGTGTGTGACTG	129	60
eIF-2α	XM_001928339.4	ATCAGCACAAATTTCCTGAGGT TGCCTTCGATGTTGTTGTATTC	108	58
ATF4	NM_001123078.1	GTGGAGAAAATGGATCTGAAGG CAACCTGGTCAGTTGTTGGTAA	196	59
β-actin	XM_003124280.5	AGATCACTCCCCCAATGACAG AGAGCAAGAGAGGCATCCTG	111	58

Table 3. The PCR primers used to amplify the genes encoding PKB, 4E-BP1, S6K1, eIF-2α, ATF4 and β-actin in cattle.

Target genes	Reference sequence	Primer sequence (5'→3')	Product size (bp)	Annealing temperature(°C)
PKB	NM_173986.2	GTTTGAGTACGTGAAGCTGCTG CACGATGACCTCCTTCTTTAGG	115	59
4E-BP1	NM_001077893.2	AGGTAGCAGCTGCAGCCAGACC GCTTGTCGTCTGGGCTACTG	292	60
S6K1	NM_205816.1	ATTCATGACGGAACAGTCACAC AGGGAGGCAAATTGAGTTTACA	214	59
eIF-2α	NM_175813.2	GCTGGAATACAACAACATCGAA ACATTTGATTGCTTCCTCTGGT	187	60
ATF4	NM_001034342.2	GCTACTAGGTACCGCCAGAAGA GGAATCTGCCTTCTCTTTCAGA	105	60
β-actin	NM_173979.3	CGGCATTCACGAAACTACCT GATCTCTTTCTGCATCCTGTCC	136	59

Table 4. Interaction of Leu on pancreatic enzyme activity of different animals in vitro.

Animal Leu (mg/mL)	cattle				Pig				SEM^a	P-value^b
	0	2.62	5.24	10.48	0	2.62	5.24	10.48		Animal	Leu	Interaction
Tissue
α-amylase (IU/mg)	44	51	56	72	28	30	34	53	0.01	0.07	0.001	0.85
Lipase (IU/g)	0.25	0.26	0.29	0.29	0.18	0.19	0.27	0.32	0.05	0.01	0.07	0.80
Trypsin (IU/g)	4245	4313	6420	6565	2221	2480	2209	2599	271.40	<.001	<.001	0.004
Chymotrypsin (IU/g)	1.69	3.78	8.34	8.26	2.38	3.32	6.46	5.59	0.33	0.21	0.003	<.001
Release
α-amylase (IU/g)	760	798	1010	965	624	703	928	962	62.08	0.09	<.001	0.72
Lipase (IU/g)	1.32	1.34	1.37	1.69	1.75	1.98	1.96	2.17	0.07	<.001	0.61	<.001
Trypsin (IU/g)	18000	15257	17127	17033	12030	14073	13512	20088	1289.14	<.001	<.001	0.007
Chymotrypsin (IU/g)	15.62	16.60	21.81	20.03	26.16	33.63	31.32	39.54	0.45	<.001	<.001	<.001

^a Pooled standard error of the mean, n = 5.

^b Differences were considered significant at P < 0.05.

Figure 1. The effect of Leu on the expression of key genes in the mTOR pathway in the pancreatic tissue of cattle and pigs in vitro.

Figure 2. The effect of Leu on the expression and phosphorylation of key proteins in the mTOR signaling pathway in the pancreatic tissue of cattle and pigs in vitro.

Figure 3. The effect of Leu on the expression of key genes in the ISR pathway in the pancreatic tissue of cattle and pigs in vitro.

Figure 4. The effect of Leu on the expression and phosphorylation of key proteins in the ISR signaling pathway in the pancreatic tissue of cattle and pigs in vitro.

Data availability statement

The data used to support the findings of this study are available from the corresponding author upon request.