Table 1.
List of primers used for qPCR.
Table 2.
Gavage-amino acid mixture.
Fig 1.
Axial expression and localization of B0AT1, ACE2 and CD13.
A: Localization of B0AT1, ACE2 and CD13 by immunofluorescence analysis. Expression of B0AT1, ACE2 and CD13 in the ileum is visualized by confocal microscopy. Upper panel: low-and high magnification images of protein of interest; lower panel: low-magnification images of protein of interest (green) and DAPI (blue). B: Relative mRNA abundance of B0AT1 and ACE2 in duodenum versus ileum. The mRNA expression levels of B0AT1 and ACE2 were measured by quantitative real-time PCR. Rats were either euthanized 3 h after light-onset (ZT3) or 3 h after light-offset (ZT15). Scraped mucosa of duodenum and ileum was used for analysis. The level of tested mRNAs standardized to 18S rRNA and normalized to the ZT15 value in the ileum is shown. The values represent mean levels from 27 rats pooled from three independent experiments in which rats were fed either a NP, HP or AA diet. Values are expressed as means ± SEM; n = 27; two-way ANOVA and Bonferroni's post test; *** P < 0.001, ** P < 0.01. C: Protein expression of B0AT1, ACE2 and CD13 along the small intestine. Western blotting experiments of bbmv with antibodies directed against B0AT1, ACE2, CD13 and β-actin were performed. Representative Western blotting images are shown in the left panels; D = duodenum, JP = proximal jejunum, JM = middle jejunum, JD = distal jejunum, I = ileum; Molecular weight markers are indicated in kDa. The intensity of the immunoreactive bands was quantified, standardized to β-actin and normalized for each time point to JP in order to highlight the differences between JP and JD. Data shown in the right panels represent mean levels from 18–27 rats pooled from three independent experiments in which rats were fed either a NP, HP or AA diet. Values are expressed as means ± SEM; n = 18–27; two-way ANOVA and Bonferroni's post test; *** P < 0.001.
Fig 2.
Circadian effect on B0AT1 protein expression and L-isoleucine uptake rate.
Animals were fed NP, HP or AA diet for 7 days and euthanized either 3 h after light-onset (ZT3) or 3 h after light-offset (ZT15). Western blotting experiments with antibodies directed against B0AT1 and β-actin were performed with bbmv of the proximal and distal jejunum (same preparations as in Fig 1) and the intensity of the immunoreactive bands was quantified, standardized to β-actin and normalized to ZT3 in order to highlight the differences between time points. Data represent mean values of 9 different animals. Values are expressed as means ± SEM, two-way ANOVA and Bonferroni's post test; *** P < 0.001. The intestinal ring-uptake of L-isoleucine in proximal and distal jejunum at ZT3 and ZT15 was measured in the presence and absence of sodium and sodium-dependent uptake was calculated. Each uptake condition was measured in triplicates. Data represent mean values of 9 different animals from 3 independent experiments. Values are expressed as means ± SEM, two-way ANOVA and Bonferroni's post test, ** P < 0.01, *** P < 0.001.
Fig 3.
Impact of an amino acid cocktail on the time course of gastric emptying.
Food-deprived rats were gavaged with contrast agent (sodium diatrizoate hydrate, SDH) and water or an isomolar amino acid cocktail and the impact on gastric emptying was measured with computed tomography (CT). Three-dimensional volume renderings of representative stomachs are shown immediately (0 min), 20, 40 and 60 min after gavage (A). Gastric SDH content was quantified over time which reflects the gastric emptying (B). Values are expressed as means ± SEM; n = 5.
Fig 4.
Lack of short-term effect by amino acid cocktail gavage on L-isoleucine transport and B0AT1 membrane expression.
Food-deprived rats were gavaged with water or an isomolar amino acid cocktail. One hour after gavage, the transport of L-isoleucine into everted rings of the middle jejunum was measured in the presence (black bars) or absence of sodium (white bars) (A). Data represent mean values of 5 rats ± SEM, each uptake condition was measured in triplicates; no significant difference of sodium-dependent uptake by paired two-tailed Students t-test. B: Western blotting experiments of bbmv with antibodies directed against B0AT1 and β-actin were performed. Representative Western blotting images are shown. Molecular weight markers are indicated in kDa.
Fig 5.
Effect of different diets on L-isoleucine transport and B0AT1, ACE2 and CD13 membrane expression.
Animals fed NP, HP or AA diet for 7 days were euthanized 3 h after light-onset (ZT3) or offset (ZT15). A: The transport of L-isoleucine into everted rings of the proximal and distal jejunum was measured in the presence and absence of sodium and sodium-dependent uptake was calculated. Data represent mean values of 3 intestinal ring uptakes of 9 different animals tested in 3 independent experiments. B-D: Western blotting experiments with antibodies directed against B0AT1, ACE2, CD13 and β-actin were performed in bbmv of the proximal and distal jejunum (same preparations as in Fig 1). The intensity of the immunoreactive bands was quantified, standardized to β-actin and normalized to NP diet. Data represent mean values of 9 different animals. All values are expressed as means ± SEM, one-way ANOVA and Bonferroni’s post test was performed for each time point; * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 6.
Effect of different diets on L-isoleucine absorption along the small intestine following gavage.
Animals were fed NP, HP or AA diet for 7 days. An amino acid mixture (Table 2) supplemented with radiolabeled L-isoleucine was gavaged 3 h after light offset and after 1 h, the intestinal content of different segments was flushed, mucosa was scraped and blood (plasma) collected to measure radiolabeled L-isoleucine (A—C). Plasma concentrations of L-isoleucine, L-leucine, L-valine, glycine, L-serine and L-threonine were determined by UPLC. Data represent mean values of 9 different animals per group, values are expressed as means ± SEM, one-way ANOVA and Bonferroni’s post test for each amino acid; * P < 0.05, ** P < 0.01, *** P < 0.001.