Apical GLUT2 and Cav1.3: regulation of rat intestinal glucose and calcium absorption

Abstract

We have proposed a model of intestinal glucose absorption in which transport by SGLT1 induces rapid insertion and activation of GLUT2 in the apical membrane by a PKC betaII-dependent mechanism. Since PKC betaII requires Ca(2+) and glucose is depolarizing, we have investigated whether glucose absorption is regulated by the entry of dietary Ca(2+) through Ca(v)1.3 in the apical membrane. When rat jejunum was perfused with 75 mM glucose, Ca(2+)-deplete conditions, or perfusion with the L-type antagonists nifedipine and verapamil strongly diminished the phloretin-sensitive apical GLUT2, but not the phloretin-insensitive SGLT1 component of glucose absorption. Western blotting showed that in each case there was a significant decrease in apical GLUT2 level, but no change in SGLT1 level. Inhibition of apical GLUT2 absorption coincided with inhibition of unidirectional (45)Ca(2+) entry by nifedipine and verapamil. At 10 mM luminal Ca(2+), (45)Ca(2+) absorption in the presence of 75 mM glucose was 2- to 3-fold that in the presence of 75 mM mannitol. The glucose-induced component was SGLT1-dependent and nifedipine-sensitive. RT-PCR revealed the presence of Ca(v)beta(3) in jejunal mucosa; Western blotting and immunocytochemistry localized Ca(v)beta(3) to the apical membrane, together with Ca(v)1.3. We conclude that in times of dietary sufficiency Ca(v)1.3 may mediate a significant pathway of glucose-stimulated Ca(2+) entry into the body and that luminal supply of Ca(2+) is necessary for GLUT2-mediated glucose absorption. The integration of glucose and Ca(2+) absorption represents a complex nutrient-sensing system, which allows both absorptive pathways to be regulated rapidly and precisely to match dietary intake.

Figure 1. The effect of luminal Ca²⁺ and L-type effectors on glucose absorption

A, a representative time course showing the effect of Ca²⁺. Rat mid and distal jejunum was pre-perfused in vivo in single-pass mode with modified KHB + 100 mm mannitol containing either 1.25 mm Ca²⁺ (▪) or no Ca²⁺ (□, Ca²⁺-deplete perfusate); after 20 min the perfusate was switched to 75 mm glucose + 25 mm mannitol to achieve an initial steady state (30–50 min) before the addition of 1 mm phloretin at 50 min (arrow) to determine the phloretin-insensitive (SGLT1) steady-state rate (60–90 min). B, the effect of L-type effectors, which were present from the start of perfusions. Total (black bars) and phloretin-insensitive (hatched bars) steady-state rates were obtained for control, Ca²⁺ deplete, nifedipine (10 μm, Nif) and verapamil (100 μm, Ver) perfusions (n=4, each case). Rates are means ± s.e.m. expressed in μmol min⁻¹ (g dry wt)⁻¹. Student's t test was used to determine statistical significance. †††P < 0.001, unpaired test for the comparison of the initial steady-state rates of the Ca²⁺ deplete, nifedipine and verapamil to that of the control. ***P < 0.001, **P < 0.01, *P < 0.05, paired test comparing the initial steady-state rate with its phloretin-insensitive rate. The phloretin-insensitive rates are not statistically different from one another.

Figure 2. Inhibition of glucose absorption occurs concomitantly with inhibition of ⁴⁵Ca²⁺ absorption by L-type channel antagonists

Jejunum was perfused in vivo with perfusate comprising modified KHB containing 75 mm glucose, 1.25 mm Ca²⁺ and ⁴⁵Ca²⁺ (0.35 kBq ml⁻¹) as a tracer to determine unidirectional lumen-to-mucosa ⁴⁵Ca²⁺ absorption. After a control period, the perfusion was switched (arrow) to an experimental period in which perfusate contained an L-type antagonist. Time courses are shown for: (A) the rate of glucose absorption when (C) ⁴⁵Ca²⁺ absorption was inhibited by nifedipine (10 μm) and for (B) the rate of glucose absorption when (D) ⁴⁵Ca²⁺ absorption was inhibited by verapamil (100 μm). Absorption rates are presented as μmol min⁻¹ (g dry wt)⁻¹ and means ± s.e.m., n=4.

Figure 3. Dependence of GLUT2, PKC βII and SGLTI levels on luminal Ca²⁺ and nifedipine

Brush-border membrane vesicles were prepared from rat jejunum initially perfused in vivo with 75 mm glucose in modified KHB (1.25 mm Ca²⁺). At 20 min, perfusates were switched to 75 mm glucose in KHB containing either 1.25 mm Ca²⁺ (control), 0 mm Ca²⁺ (Ca²⁺ deplete), or 1.25 mm Ca²⁺ containing nifedipine (10 μm). Vesicle protein (20 μg) was then separated by SDS-PAGE (10% gels), transblotted on to PVDF and Western blotted for GLUT2, SGLT1 and PKC βII. For full details, see Methods.

Figure 4. Activation of ⁴⁵Ca²⁺ absorption by glucose

A, the rates of ⁴⁵Ca²⁺ absorption in the presence of either mannitol or glucose were compared within a single perfusion (paired comparison): rat jejunum was perfused for a control period of 40 min with 75 mm glucose and 10 mm Ca²⁺ (n=4). After 40 min (arrow), perfusion was continued for an experimental period in which 75 mm mannitol was substituted for 75 mm glucose. B, separate perfusions (unpaired comparison): rat jejunum was perfused with 75 mm glucose in the presence of 10 mm Ca²⁺ for 40 min only (n=8); separate perfusions were undertaken in which mannitol replaced glucose (n=8). P < 0.001 for comparison of the effects of glucose and mannitol in A and B (†††).

Figure 5. Ca²⁺ absorption at high Ca²⁺ concentration also displays L-type characteristics

A and C, jejunum was perfused for a control period of 40 min with 75 mm glucose and 10 mm Ca²⁺ (n=4). After 40 min (arrow), perfusion was continued for an experimental period with 1 mm phloridzin also present in the perfusate. A and C show the glucose and Ca²⁺ fluxes, respectively. B and D, jejunum was perfused for a control period of 40 min with 75 mm glucose and 10 mm Ca²⁺ (n=4). After 40 min (arrow), perfusion was continued for an experimental period with 10 μm nifedipine also present in the perfusate. B and D show the glucose and Ca²⁺ fluxes, respectively. Rates are presented as μmol min⁻¹ (g dry wt)⁻¹.

Figure 6. Rat jejunal mucosa expresses β₃ mRNA and β₃ protein is located in the apical membrane

A, RT-PCR was performed for the four known auxiliary Ca_vβ subunits using primers designed to rat specific sequences and cDNA derived from rat brain and rat jejunal mucosa. B, jejunum was perfused for 30 min with 75 mm glucose; brush-border membrane (BBM) and basolateral membrane (BLM) vesicles were then prepared and Western blotted for β₃. The BBM band was eliminated by neutralization of antibody with excess peptide (data not shown).

Figure 7. Immunocytochemical localization of Ca_v1.3 and β₃-subunit in the proximal jejunum

Sections of unperfused jejunum were labelled with a rabbit antibody that recognizes all forms of rat Ca_v1.3 (A and B) and with a rabbit antibody which specifically recognizes the β₃ subunit (C and D). The secondary antibody was FITC-conjugated goat anti-rabbit IgG. The peptide controls were sections treated with antibody to Ca_v1.3 (B) and β₃ (D) that had been pre-absorbed with excess antigenic peptide; non-specific staining in the lamina propria of these sections was seen with the secondary alone. All sections are at 63 × magnification and were taken at the same settings with a Zeiss LSM 510 confocal microscope.