CCK and GLP-1 release in response to proteinogenic amino acids using a small intestine ex vivo model in pigs

Abstract

The impact of individual amino acids (AA) on gut hormone secretion and appetite regulation in pigs remains largely unknown. The aim of the present study was to determine the effect of the 20 proteinogenic AA on the release of the anorexigenic hormones cholecystokinin (CCK) and glucagon-like peptide 1 (GLP-1) in postweaning pigs. Six 25-d-old male piglets (Domestic Landrace × Large White; body weight = 6.94 ± 0.29 kg) were humanely killed for the collection of intestinal segments from the duodenum, jejunum, and ileum. Tissue samples from the three intestinal segments were used to determine which of the regions were more relevant for the analysis of gut peptides. Only the segments with the highest CCK and GLP-1 secretion and expression levels were evaluated with the 20 individual AA. Tissue segments were cut open, cleaned, and stripped of their muscle layer before identical circular samples were collected and incubated in 24-well plates for 1 h (37 °C, 5% v/v CO2). The culture broth consisted of a glucose-free KRB buffer containing no added AA (control) or with the addition of 10 mM of 1 of the 20 proteinogenic AA. Following incubation, tissues and supernatant were collected for gene expression and secretion analysis of CCK and GLP-1 levels. CCK secretion and mRNA expression were higher (P < 0.05) in duodenum when compared with proximal jejunum or ileum, whereas GLP-1/proglucagon levels were higher in ileum vs. duodenum (P < 0.05) and jejunum (P < 0.05, for GLP-1 only) in postweaning pigs. Based on these results, the effect of AA on CCK and GLP-1 secretion was studied in the duodenum and ileum, respectively. None of the AA tested stimulated both anorexigenic hormones. Of all the essential AA, Ile, Leu, Met, and Trp significantly (P < 0.05) stimulated GLP-1 from the ileum, while only Phe stimulated CCK from the duodenum. Of the nonessential AA, amide AA (Gln and Asn) caused the release of CCK, while Glu and Arg increased the release of GLP-1 from the ileum. Interpreting the results in the context of the digestion and absorption dynamics, non-bound AA are quickly absorbed and have their effect on gut peptide secretion limited to the proximal small intestine (i.e., duodenum), thus, mainly CCK. In contrast, protein-bound AA would only stimulate CCK release from the duodenum through feedback mechanisms (such as through GLP-1 secreted mainly in the ileum).

Keywords: amino acid; cholecystokinin; gastrointestinal tract; glucagon-like peptide 1; pig.

Figure 1.

Cholecystokinin (CCK) secretion and gene expression levels along the small intestine of weaner pigs. Tissue hormone levels (measured in supernatant) (A) and mRNA expression (B) of CCK in primary cultures of duodenum (Duo), proximal jejunum (P. Jeju), and ileum (Ile) of postweaning pigs incubated for 1 h in KRB/HEPES free glucose buffer (n = 6). Data are expressed as the mean + SEM. Different letters (a, b, and c) indicate significant differences (P < 0.05).

Figure 2.

Glucagon-like-peptide 1 (GLP-1) secretion and proglucagon (GCG) gene expression levels along the small intestine of weaner pigs. Tissue hormone levels of GLP-1 (measured in supernatant) (A) and GCG gene expression (B) in primary cultures of duodenum (Duo), proximal jejunum (P. Jeju), and ileum (Ile) of postweaning pigs incubated for 1 h in KRB/HEPES free glucose buffer (n = 6). Data are expressed as the mean + SEM. Different letters (a and b) within the same graph indicate significant differences between tissues (P < 0.05).

Figure 3.

Duodenal cholecystokinin (CCK) secretion following essential amino acid (EAA) stimulation in weaner pigs. CCK secretion form porcine duodenum cultures following a 1 h incubation with EAA (His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) at 10 mM. Data are expressed as percentage of control (KRB buffer alone = dotted line). n = 6, each data point within boxplots represents the average of three biological replicates within each pig. *P < 0.05.

Figure 4.

Duodenal cholecystokinin (CCK) secretion following non-essential amino acid (NEAA) stimulation in weaner pigs. CCK secretion form porcine duodenum cultures following a 1 h incubation with NEAA at 10 (Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Pro, and Ser) or 2.5 mM (Tyr). Data are expressed as percentage of control (KRB buffer alone = dotted line). n = 6, each data point within boxplots represents the average of three biological replicates within each pig. *P < 0.05.

Figure 5.

Ileal glucagon-like peptide 1 (GLP-1) secretion following essential amino acid (EAA) stimulation in weaner pigs. GLP-1 secretion from porcine ileum cultures following a 1 h incubation with EAA (His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) at 10 mM. Data are expressed as percentage of control (KRB buffer alone = dotted line). n = 6, each data point within boxplots represents the average of three biological replicates within each pig. *P < 0.05, **P < 0.01.

Figure 6.

Ileal glucagon-like peptide 1 (GLP-1) secretion following non-essential amino acid (NEAA) stimulation in weaner pigs. GLP-1 secretion from porcine ileum cultures following a 1 h incubation with NEAA at 10 (Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Pro, and Ser) or 2.5 mM (Tyr). Data are expressed as percentage of control (KRB buffer alone = dotted line). n = 6, each data point within boxplots represents the average of three biological replicates within each pig. *P < 0.05, **P < 0.01.