Apical Na+-D-glucose cotransporter 1 (SGLT1) activity and protein abundance are expressed along the jejunal crypt-villus axis in the neonatal pig

Abstract

Gut apical Na(+)-glucose cotransporter 1 (SGLT1) activity is high at the birth and during suckling, thus contributing substantially to neonatal glucose homeostasis. We hypothesize that neonates possess high SGLT1 maximal activity by expressing apical SGLT1 protein along the intestinal crypt-villus axis via unique control mechanisms. Kinetics of SGLT1 activity in apical membrane vesicles, prepared from epithelial cells sequentially isolated along the jejunal crypt-villus axis from neonatal piglets by the distended intestinal sac method, were measured. High levels of maximal SGLT1 uptake activity were shown to exist along the jejunal crypt-villus axis in the piglets. Real-time RT-PCR analyses showed that SGLT1 mRNA abundance was lower (P < 0.05) by 30-35% in crypt cells than in villus cells. There were no significant differences in SGLT1 protein abundances on the jejunal apical membrane among upper villus, middle villus, and crypt cells, consistent with the immunohistochemical staining pattern. Higher abundances (P < 0.05) of total eukaryotic initiation factor 4E (eIF4E) protein and eIE4E-binding protein 1 γ-isoform in contrast to a lower (P < 0.05) abundance of phosphorylated (Pi) eukaryotic elongation factor 2 (eEF2) protein and the eEF2-Pi to total eEF2 abundance ratio suggest higher global protein translational efficiency in the crypt cells than in the upper villus cells. In conclusion, neonates have high intestinal apical SGLT1 uptake activity by abundantly expressing SGLT1 protein in the epithelia and on the apical membrane along the entire crypt-villus axis in association with enhanced protein translational control mechanisms in the crypt cells.

Fig. 1.

Initial rates of d-glucose uptake under a Na⁺ gradient into the apical membrane vesicles prepared from epithelial cells isolated along the jejunal crypt-villus axis in the liquid formula-fed neonatal pigs. Apical membrane vesicles were preloaded with a buffer containing 180 mM d-mannitol, 150 mM KSCN, 10 mM Trizma·HCl at pH 7.4. Uptake buffer contained 2.4 μM d-[6-³H]glucose, 180 mM d-mannitol, 150 mM NaSCN, and 10 mM Trizma·HCl at pH 7.4. The uptake media (60 μl), resulting from mixing 50 μl uptake buffer with 10 μl apical membrane vesicles, contained 2.0 μM d-[6-³H]glucose, 180 mM d-mannitol, 125 mM NaSCN, 25 mM KSCN, and 10 mM Trizma·HCl. Each point represents mean and SE (n = 3) of measurements from 3 uptake experiments (duplicate observations in each experiment) using 3 separate batches of apical membrane vesicle suspension prepared from 12 epithelial cell fractions (F1 through F12). Each batch of epithelial cell fractions was collected and pooled from the proximal and the distal jejunal segments of 2 piglets; thus a total of 6 piglets were used in these uptake experiments. J, rate of d-[6-³H]glucose tracer uptake into apical membrane vesicles.

Fig. 2.

Kinetics of sodium-d-glucose cotransporter 1 (SGLT1) glucose uptake into the apical membrane vesicles prepared from the upper villus (A), the middle villus (B), and the crypt epithelial cells (C) isolated along the jejunal crypt-villus axis in the neonatal pig. Initial rates of the d-[6-³H]glucose tracer uptake as a function of extravesicular concentrations of nonlabeled d-glucose as shown. Apical membrane vesicles were preloaded with a buffer containing 180 mM d-mannitol, 150 mM KSCN, 10 mM Trizma·HCl at pH 7.4. A total of 19 uptake buffers contained 2.4 μM d-[6-³H]glucose, 150 mM NaSCN, and 10 mM Trizma·HCl at pH 7.4 and differed in the concentrations of d-glucose (0–110 mM) and d-mannitol (180–70 mM). The uptake media (60 μl), resulting from mixing 50 μl uptake buffer and 10 μl apical membrane vesicles, contained 2.0 μM d-[6-³H]glucose, 125 mM NaSCN, 25 mM KSCN, 0–92 mM d-glucose, 180–88 mM d-mannitol, and 10 mM Trizma·HCl. Each point represents mean and SE (n = 3) of measurements from 3 uptake experiments (duplicate observations in each experiment) using 3 separate batches of apical membrane vesicle suspension prepared from 3 epithelial cell fractions (the upper villus cell, F1–F4; the middle villus cell, F5–F8; and the crypt cell, F9–F12). Each batch of epithelial cell fractions was collected and pooled from the proximal and the distal segments of two piglets; thus a total of 18 piglets were used in these experiments. J_max, maximal rate of d-glucose uptake into the apical membrane vesicles; K_m, the SGLT1 affinity; J_diffusion, apparent transmembrane diffusion rate of d-glucose into the apical membrane vesicles; S_cold, extracellular concentration of unlabeled d-glucose.

Fig. 3.

Real-time RT-PCR analysis of SGLT1 mRNA abundances in the upper villus (U), the middle villus (M), and crypt (C) epithelial cells isolated along the jejunal crypt-villus axis in the liquid formula-fed neonatal pigs. Results were normalized using β-actin expression as a housekeeping control gene in each real-time RT-PCR. Data are expressed relative to the crypt cell value (value = 1) and are presented as means ± SE (n = 6) in arbitrary units. Bars with a different superscript letters differ (P < 0.05).

Fig. 4.

Western blot analysis of SGLT1 protein abundances in the upper villus, the middle villus, and crypt epithelial cells isolated along the jejunal crypt-villus axis in the liquid formula-fed neonatal pigs. Data are in arbitrary units and are presented as means ± SE (n = 6).

Fig. 5.

Immunohistochemical localization of SGLT1 along the jejunal crypt-villus axis in the liquid formula-fed neonatal pigs. Expression of SGLT1 (brown) was observed on the apical membrane along the entire crypt-villus axis in both the proximal (A) and the distal (B) jejunal regions (left). The staining was not observed when the primary antibody was excluded (right). All images are shown at ×10 magnification.

Fig. 6.

Western blot analysis of eukaryotic initiation factor 4E (eIF4E) protein abundances in the upper villus, the middle villus, and crypt epithelial cells isolated along the jejunal crypt-villus axis in the liquid formula-fed neonatal pigs. Data were normalized with β-actin as the housekeeping control gene protein in arbitrary units and are presented as means ± SE (n = 6). Bars with different superscript letters differ (P < 0.05).

Fig. 7.

Western blot analysis of eIF4E-binding protein 1 (eIF4E-BP1) γ-isomer abundances in the upper villus, the middle villus, and crypt epithelial cells isolated along the jejunal crypt-villus axis in the liquid formula-fed neonatal pig. Data were normalized with β-actin as the housekeeping protein in arbitrary units and are presented as means ± SE (n = 6). Bars with different superscript letters differ (P < 0.05).

Fig. 8.

Western blot analysis of total eukaryotic elongation factor 2 (eEF2) protein abundances in the upper villus, the middle villus, and crypt epithelial cells isolated along the jejunal crypt-villus axis in the liquid formula-fed neonatal pig. Data were normalized with β-actin as the housekeeping protein in arbitrary units and are presented as means ± SE (n = 6).

Fig. 9.

Western blot analysis of phosphorylated eEF2 (phospho-eEF2) protein abundances in the upper villus, the middle villus, and crypt epithelial cells isolated along the jejunal crypt-villus axis in the liquid-formula-fed neonatal pig. Data were normalized with β-actin as the housekeeping protein in arbitrary units and are presented as means ± SE (n = 6). Bars with different superscript letters differ (P < 0.05).

Fig. 10.

Phospho-eEF2 protein abundance to total eEF2 protein abundance ratio in the upper villus, the middle villus, and crypt epithelial cells isolated along the jejunal crypt-villus axis in the liquid formula-fed neonatal pigs. Data are presented as means ± SE (n = 6). Bars with a different superscript letters differ (P < 0.05).