Study On The Hydrolysis And Absorption Of Glutaminate-containing Dipeptides And Their Effects On The Jejunum Development Of Weaned Piglets

Three experiments were conducted to study the hydrolysis and absorption characteristics of Ala-Gin and Gly-Gln dipeptide in small intestine, and the effects of Ala-Gln and Gly-Gln dipeptide on jejunum development of weaned piglets.Exp.1. Hydrolysis characteristics of glutamine-containing dipeptides incubated with jejunum of weaned piglet in vitro.This experiment was conducted to study hydrolysis characteristics of Ala-Gln and Gly-Gln in jejunum of weaned piglets by detecting the dipeptide in culture solution. The culture soulutions were divided into three treatments, Control treatment, Ala-Gln treatment and Gly-Gln treatment, with three replicates per treatment. The jejunal segments were incubated at 37℃for 60 min. Solution samples were taken out every 5 min. The results showed that at the end of the incubation time, the concentration of Ala-Gln dipeptide and Gly-Gln dipeptide were 9.00±1.57 mmol/L,8.85±1.78 mmol/L respectively, with no remarkable difference between the two treatments(P＞0.05). The hydrolysis rates of the two dipeptides were 0.17±0.03 mmol/L/min,0.19±0.03 mmol/L/min. The hydrolysis rate of Ala-Gln was not significantly higher than that of Gly-Gln (P>0.05). The results indicated that the content of Ala-Gln dipeptide and Gly-Gln dipeptide are gradually decreased as time extending within 60 min, but neither of them are totally hydrolyzed in the incubation solutions. This experiment gives us the suggestion that the dipeptides can be absorbed as a complete form.Exp.2. Absorption characteristics of glutamine-containing dipeptides in everted jejunum sacs of weaned piglets.This experiment was conducted to study absorption characteristics of Ala-Gln and Gly-Gln in everted jejunum sacs of weaned piglets by detecting the dipeptide and glutaminate monomer in culture solution, serosal fluid and tissue of cultured reversal intestine segments in vitro. The results showed that both Ala-Gln dipeptide and Gly-Gln dipeptide were detected in the incubated jejunal sacs tissue and serosal fluid. The content of Ala-Gln dipeptide absorbed into the sacs was 10.26±0.74 mmol/L, significantly greater than that of Gly-Gln dipeptide, 7.25±1.47 mmol/L (P<0.05). The content of Gln absorbed in Ala&Gln treatment and in Gly&Gln treatment was 7.28±0.54 mmol/L,4.79±0.5.4 mmol/L respectively, with no significant difference between the two treatments (P>0.05). Absortion content of dipeptides were significantly greater than that of Gln in relative free amino acids treatments. The absorption rate of dipeptides or Gln monomer had the same changing characteristics as the absortion content of dipeptides or Gln monomer. The results indicated that Ala-Gln and Gly-Gln were absorbed by intestinal cells as intact form, and quickly hydrolyzed inside the cells. The absorption rate of Ala-Gln was faster than that of Gly-Gln. Dipeptides absorption rate were faster than Gln absorption rate in free form.Exp.3. Effects of glutamine-containing dipeptides on jejunum development of weaned piglets.This experiment was conducted to study effects of Ala-Gln dipeptide and Gly-Gln dipeptide on jejunum develpoment of weaned piglets by detecting the enzyme activity, including LDH, DAO, glutaminase, and the content of BrdU incorporating into cell nuclear, the expression of Caspase-3 in vitro. Three test teams were designed, the blank control team,Ala-Gln dipeptide team and the Gly-Gln dipeptide team. There were 2 mmol/L,4 mmol/L,10 mmol/L,20 mmol/L and 30 mmol/L dipeptide concentration groups in both Ala-Gln dipeptide team and the Gly-Gln team. The intestinal tissues were cultured in CO₂ gas incubator for 48 h or 96 h. The results showed that both Ala-Gln dipeptide and Gly-Gln dipeptide down regulated LDH activity in the culture solution. The tissue protein content, diamine oxidase activity and glutaminase activity significantly increased in both Ala-Gln dipeptide team and the Gly-Gln team, compared to the black control (P<0.05). When the concentration of Ala-Gln dipeptide and Gly-Gln dipeptide were 20 mmol/L and 30 mmol/L, respectively, the content of tissue protein and the activity of diamine oxidase were the highest. When the concentration of both dipeptides were 20 mmol/L, the activity of glutaminase reached the top. On the other hand, both dipeptide improved the percentage of BrdU-positive cell nuclei (P＜0.01), and the mean optical density of positive cells stained with anti-BrdU antibody (P<0.01). Both the dipeptides concentration reached the level of 30 mmol/L, the percentage of BrdU-positive cell nuclei and the mean optical density of positive cells reached the top. And there were no significent differences between the two dipeptides treatment (P>0.05). After incubated for 96 h, intestinal tissue began to express Caspase-3. Ala-Gln dipeptide and Gly-Gln dipeptide notably depressed the Caspase-3 expression (P<0.01). Further more, the percentage of Caspase-3 positive cells and the mean optical density of positive cells were significantly decreased in Ala-Gln dipeptide and in, Gly-Gln dipeptide teams (P<0.01), compared to the blank control. But there were no differences between the 20 mmol/L dipeptide treatment and the 30 mmol/L dipeptide treatment in both two dipeptide groups (P>0.05). The effect of both dipeptides on the enzyme activity, tissue protein content, cell proliferation and apoptosis changed along with the dipeptides concentration changing. The results indicated that either Ala-Gin dipeptide or Gly-Gln dipeptide can enchance the consumption of Gin by increasing the activities of Gin metabolic-related enzymes, thereby promote the cell proliferation, depressed cell apoptosis, and then elevate intestinal tissue protein content. The effects of both dipeptides on above detected targets are in dose-dependent effect, and the two dipeptides have similar effects at the same concentration level. The tissue glutaminase activity reach the top when the two dipeptides concentration are at 20 mmol/L. The tissue DAO activity and protein content-are the highest when the concentration of Ala-Gln is 20 mmol/L, while the contentration of Gly-Gln is 30 mmol/L. The effects of the dipeptides on cell proliferation and apoptosis are excellent at 30 mmol/L content.In conclusion, Ala-Gln and Gly-Gln dipeptides, incubated with jejunum segments of weaned piglets, are not totally hydrolyzed and their hydrolysis rate are 0.17±0.03 mmol/L/min, 0.19±0.03 mmol/L/min, respectively. Either Ala-Gln or Gly-Gln is absorbed as complete forms, and the absorption content within 40 min are 10.26±0.74 mmol/L,7.25±1.47 mmol/L. And the absorption rate of Ala-Gin is faster than Gly-Gln, both are faster than the rate of free Gln. The two dipeptides effectively promote the enzym activities, protein content of jejunal tissue, promote the cell proliferation, and depress cell apoptosis. All of the effects are in dose-dependent effect, and the two dipeptides have similar effects at the same concentration level.