Influence of fed-fasted state on intestinal PEPT1 expression and in vivo pharmacokinetics of glycylsarcosine in wild-type and Pept1 knockout mice

Abstract

Purpose: To determine if fasting would affect the intestinal expression and in vivo functional activity of PEPT1 as determined after oral dosing of the dipeptide glycylsarcosine (GlySar).

Methods: Systemic exposure and tissue distribution studies were performed in wild-type and Pept1 knockout mice, under fed and fasted conditions, following both intravenous and oral doses of [(14)C]GlySar at 5 nmol/g body weight. Intestinal PEPT1 expression was evaluated by real-time PCR and immunoblot analyses.

Results: We found that expression of PEPT1 protein in the small intestine was increased ~2-fold in wild-type mice during fasted as compared to fed conditions. In agreement, systemic exposure and peak plasma concentrations of orally administered GlySar were 40 and 65% greater, respectively, in wild-type mice during fasted vs. fed state. No significant differences were observed between fed and fasted animals during PEPT1 ablation. Tissue distribution of GlySar was unchanged after oral dosing for all four treatment groups.

Conclusions: As little as 16 h of fasting can cause significant upregulation of PEPT1 protein expression in the small intestine, which then translates into a significant increase in in vivo oral absorption of GlySar.

Figure 1

PEPT1 mRNA expression in small intestinal and colonic segments of wild-type (+/+) mice during fed and fasted conditions. mRNA expression in the duodenum of fed mice was considered the control group and was arbitrarily assigned a value of unity. mRNA expression in other treatment groups was scaled to the control value. Data are reported as mean ± SE (n=6). Statistical differences between fed and fasted conditions for each tissue segment were determined using a two-sample student's t-test; *p ≤ 0.05.

Figure 2

PEPT1 protein expression in small intestinal and colonic segments of wild-type mice during fed and fasted conditions. For each tissue, protein expression in fed mice was considered the control group and was arbitrarily assigned a value of unity. Protein expression in fasted mice was scaled to the control value. Data are reported as mean ± SE (n=6). Statistical differences between fed and fasted conditions for each tissue segment were determined using a two-sample student's t-test; **p ≤ 0.01 and ***p ≤ 0.001. Relative protein expression levels in small intestinal and colonic segments have been reported previously (see reference 5, Fig 7).

Figure 3

Plasma concentration-time profiles of [¹⁴C]GlySar during fed and fasted conditions in wild-type (+/+) and Pept1 knockout (-/-) mice after 5 nmol/g intravenous bolus doses of dipeptide. Data are reported as mean ± SE (n=6).

Figure 4

Tissue distribution of [¹⁴C]GlySar during fed and fasted conditions in wild-type (+/+) and Pept1 knockout (-/-) mice, 120 min after 5 nmol/g intravenous bolus doses of dipeptide. Data are reported as mean ± SE (n=6). One-way ANOVA and Tukey method of multiple comparisons were performed to test for differences among treatment groups: ^ARepresents significant differences between fed and fasted conditions in wild-type mice; ^Brepresents significant differences between fed and fasted conditions in Pept1 knockout mice; ^Crepresents significant differences between wild-type and Pept1 knockout mice in fed conditions; and ^Drepresents significant differences between wild-type and Pept1 knockout mice in fasted conditions.

Figure 5

Plasma concentration-time profiles of [¹⁴C]GlySar during fed and fasted conditions in wild-type (+/+) and Pept1 knockout (-/-) mice after 5 nmol/g oral doses of dipeptide. Data are reported as mean ± SE (n=6).

Figure 6

Tissue distribution of [¹⁴C]GlySar during fed and fasted conditions in wild-type (+/+) and Pept1 knockout (-/-) mice, 360 min after 5 nmol/g oral doses of dipeptide. Data are reported as mean ± SE (n=6). No statistical differences were observed using one-way ANOVA.

Figure 7

Small intestinal transit, as determined at 30 min by a charcoal meal, during fed and fasted conditions in wild-type (+/+) and Pept1 knockout (-/-) mice. Data are reported as mean ± SE (n=6). No statistical differences were observed using one-way ANOVA.

Figure 8

Histology of the jejunum during fed conditions in wild-type (+/+) mice (a, b) and Pept1 knockout (-/-) mice (c, d). Sections were stained with hematoxylin and eosin, and examined by light microscopy. In (a) and (c), the bar = 1 mm; in (b) and (d), the bar = 100 μm.

Figure 9

Histology of the jejunum during fasted conditions in wild-type (+/+) mice (a, b) and Pept1 knockout (-/-) mice (c, d). Sections were stained with hematoxylin and eosin, and examined by light microscopy. In (a) and (c), the bar = 1 mm; in (b) and (d), the bar = 100 μm.