Alternative perspective on intestinal calcium absorption: proposed complementary actions of Ca(v)1.3 and TRPV6.

Transcellular models of dietary Ca(2+) absorption by the intestine assign essential roles to TRPV6 and calbindin-D(9K) . However, studies with gene-knockout mice challenge this view. Something fundamental is missing. The L-type channel Ca(v) 1.3 is located in the apical membrane from the duodenum to the ileum. In perfused rat jejunum in vivo and in Caco-2 cells, Ca(v) 1.3 mediates sodium glucose transporter 1 (SGLT1)-dependent and prolactin-induced active, transcellular Ca(2+) absorption, respectively. TRPV6 is activated by hyperpolarization and is vitamin D dependent; in contrast, Ca(v) 1.3 is activated by depolarization and is independent of calbindin-D(9K) and vitamin D. This review considers evidence supporting the idea that Ca(v) 1.3 and TRPV6 have complementary roles in the regulation of intestinal Ca(2+) absorption as depolarization and repolarization of the apical membrane occur during and between digestive periods, respectively, and as chyme moves from one intestinal segment to another and food transit times increase. Reassessment of current arguments for paracellular flow reveals that key phenomena have alternative explanations within the integrated Ca(v) 1.3/TRPV6 view of transcellular Ca(2+) absorption.

An energy supply network of nutrient absorption coordinated by calcium and T1R taste receptors in rat small intestine.

T1R taste receptors are present throughout the gastrointestinal tract. Glucose absorption comprises active absorption via SGLT1 and facilitated absorption via GLUT2 in the apical membrane. Trafficking of apical GLUT2 is rapidly up-regulated by glucose and artificial sweeteners, which act through T1R2 + T1R3/alpha-gustducin to activate PLC beta2 and PKC betaII. We therefore investigated whether non-sugar nutrients are regulated by taste receptors using perfused rat jejunum in vivo. Under different conditions, we observed a Ca(2+)-dependent reciprocal relationship between the H(+)/oligopeptide transporter PepT1 and apical GLUT2, reflecting the fact that trafficking of PepT1 and GLUT2 to the apical membrane is inhibited and activated by PKC betaII, respectively. Addition of L-glutamate or sucralose to a perfusate containing low glucose (20 mM) each activated PKC betaII and decreased apical PepT1 levels and absorption of the hydrolysis-resistant dipeptide L-Phe(PsiS)-L-Ala (1 mM), while increasing apical GLUT2 and glucose absorption within minutes. Switching perfusion from mannitol to glucose (75 mM) exerted similar effects. c-glutamate induced rapid GPCR internalization of T1R1, T1R3 and transducin, whereas sucralose internalized T1R2, T1R3 and alpha-gustducin. We conclude that L-glutamate acts via amino acid and glucose via sweet taste receptors to coordinate regulation of PepT1 and apical GLUT2 reciprocally through a common enterocytic pool of PKC betaII. These data suggest the existence of a wider Ca(2+) and taste receptor-coordinated transport network incorporating other nutrients and/or other stimuli capable of activating PKC betaII and additional transporters, such as the aspartate/glutamate transporter, EAAC1, whose level was doubled by L-glutamate. The network may control energy supply.

Sugar absorption in the intestine: the role of GLUT2.

Intestinal glucose absorption comprises two components. One is classical active absorption mediated by the Na+/glucose cotransporter. The other is a diffusive component, formerly attributed to paracellular flow. Recent evidence, however, indicates that the diffusive component is mediated by the transient insertion of glucose transporter type 2 (GLUT2) into the apical membrane. This apical GLUT2 pathway of intestinal sugar absorption is present in species from insect to human, providing a major route at high sugar concentrations. The pathway is regulated by rapid trafficking of GLUT2 to the apical membrane induced by glucose during assimilation of a meal. Apical GLUT2 is therefore a target for multiple short-term and long-term nutrient-sensing mechanisms. These include regulation by a newly recognized pathway of calcium absorption through the nonclassical neuroendocrine l-type channel Cav1.3 operating during digestion, activation of intestinal sweet taste receptors by natural sugars and artificial sweeteners, paracrine and endocrine hormones, especially insulin and GLP-2, and stress. Permanent apical GLUT2, resulting in increased sugar absorption, is a characteristic of experimental diabetes and of insulin-resistant states induced by fructose and fat. The nutritional consequences of apical and basolateral GLUT2 regulation are discussed in the context of Western diet, processed foods containing artificial sweeteners, obesity, and diabetes.

Sweet taste receptors in rat small intestine stimulate glucose absorption through apical GLUT2.

Natural sugars and artificial sweeteners are sensed by receptors in taste buds. T2R bitter and T1R sweet taste receptors are coupled through G-proteins, alpha-gustducin and transducin, to activate phospholipase C beta2 and increase intracellular calcium concentration. Intestinal brush cells or solitary chemosensory cells (SCCs) have a structure similar to lingual taste cells and strongly express alpha-gustducin. It has therefore been suggested over the last decade that brush cells may participate in sugar sensing by a mechanism analogous to that in taste buds. We provide here functional evidence for an intestinal sensing system based on lingual taste receptors. Western blotting and immunocytochemistry revealed that all T1R members are expressed in rat jejunum at strategic locations including Paneth cells, SCCs or the apical membrane of enterocytes; T1Rs are colocalized with each other and with alpha-gustducin, transducin or phospholipase C beta2 to different extents. Intestinal glucose absorption consists of two components: one is classical active Na+-glucose cotransport, the other is the diffusive apical GLUT2 pathway. Artificial sweeteners increase glucose absorption in the order acesulfame potassium approximately sucralose > saccharin, in parallel with their ability to increase intracellular calcium concentration. Stimulation occurs within minutes by an increase in apical GLUT2, which correlates with reciprocal regulation of T1R2, T1R3 and alpha-gustducin versus T1R1, transducin and phospholipase C beta2. Our observation that artificial sweeteners are nutritionally active, because they can signal to a functional taste reception system to increase sugar absorption during a meal, has wide implications for nutrient sensing and nutrition in the treatment of obesity and diabetes.

Calcium absorption by Cav1.3 induces terminal web myosin II phosphorylation and apical GLUT2 insertion in rat intestine.

Glucose absorption in rat jejunum involves Ca(2+)- and PKC betaII-dependent insertion of GLUT2 into the apical membrane. Ca(2+)-induced rearrangement of the enterocyte cytoskeleton is thought to enhance paracellular flow. We have therefore investigated the relationships between myosin II regulatory light chain phosphorylation (RLC(20)), absorption of glucose, water and calcium, and mannitol clearance. ML-7, an inhibitor of myosin light chain kinase, diminished the phloretin-sensitive apical GLUT2 but not the phloretin-insensitive SGLT1 component of glucose absorption in rat jejunum perfused with 75 mM glucose. Western blotting and immunocytochemistry revealed marked decreases in RLC(20) phosphorylation in the terminal web and in the levels of apical GLUT2 and PKC betaII, but not SGLT1. Perfusion with phloridzin or 75 mM mannitol, removal of luminal Ca(2+), or inhibition of unidirectional (45)Ca(2+) absorption by nifedipine exerted similar effects. ML-7 had no effect on the absorption of 10 mM Ca(2+), nor clearance of [(14)C]-mannitol, which was less than 0.7% of the rate of glucose absorption. Water absorption did not correlate with (45)Ca(2+) absorption or mannitol clearance. We conclude that the Ca(2+) necessary for contraction of myosin II in the terminal web enters via an L-type channel, most likely Ca(v)1.3, and is dependent on SGLT1. Moreover, terminal web RLC(20) phosphorylation is necessary for apical GLUT2 insertion. The data confirm that glucose absorption by paracellular flow is negligible, and show further that paracellular flow makes no more than a minimal contribution to jejunal Ca(2+) absorption at luminal concentrations prevailing after a meal.

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

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.

Affinity prediction for substrates of the peptide transporter PepT1.

A quantitative method has been developed for determining the affinity of substrates for the peptide transporter PepT1, allowing oral availability of drugs via PepT1 to be estimated.

Probing dipeptide trans/cis stereochemistry using pH control of thiopeptide analogues, and application to the PepT1 transporter.

The stereochemistry of thiodipeptides of proline [e.g. Ala-Psi[CS-N]-Pro] can be controlled using pH, allowing the trans-preference for substrates of the peptide transporter PepT1 to be confirmed.

Conformational and spacial preferences for substrates of PepT1.

The conformation at the first residue of dipeptide substrates for the peptide transporter PepT1 has been probed using constrained peptide analogues, and the active conformation has been identified.

Apical GLUT2: a major pathway of intestinal sugar absorption.

Understanding the mechanisms that determine postprandial fluctuations in blood glucose concentration is central for effective glycemic control in the management of diabetes. Intestinal sugar absorption is one such mechanism, and studies on its increase in experimental diabetes led us to propose a new model of sugar absorption. In the apical GLUT2 model, the glucose transported by the Na(+)/glucose cotransporter SGLT1 promotes insertion of GLUT2 into the apical membrane within minutes, so that the mechanism operates during assimilation of a meal containing high-glycemic index carbohydrate to provide a facilitated component of absorption up to three times greater than by SGLT1. Here we review the evidence for the apical GLUT2 model and describe how apical GLUT2 is a target for multiple short-term nutrient-sensing mechanisms by dietary sugars, local and endocrine hormones, cellular energy status, stress, and diabetes. These mechanisms suggest that apical GLUT2 is a potential therapeutic target for novel dietary or pharmacological approaches to control intestinal sugar delivery and thereby improve glycemic control.

Stress and glucocorticoid inhibit apical GLUT2-trafficking and intestinal glucose absorption in rat small intestine.

We have proposed a new model of rat intestinal sugar absorption in which high glucose concentrations promote rapid insertion of GLUT2 into the apical membrane, so that absorptive capacity is precisely regulated to match dietary intake. Construction and building work during expansion and refurbishment of our department permitted opportunistic experiments on the effects of building-induced stress on the GLUT2 component of absorption. In fed rats perfused with 75 mM glucose in vivo, stress rapidly inhibited glucose absorption 36.4 +/- 3.0% compared with control rats. Selective inhibition of the GLUT2 component with phloretin demonstrated that stress inhibited the GLUT2 component by 42.8 +/- 3.8%, which correlated with a corresponding diminution in apical GLUT2 levels: the SGLT1 component and its level were unaltered by stress. Effects of stress were reversed by the administration in drinking water of metyrapone, which inhibits 11-beta-hydroxylase. Injection of dexamethasone into control rats 60 min before perfusion resulted in absorption and transporter properties indistinguishable from stressed rats. Our data are consistent with the view that stress activates the hypothalamus-pituitary-adrenal (HPA) axis, causing release of glucocorticoid. The ensuing inhibition of GLUT2 trafficking and absorption seems necessary to prevent enhanced intestinal delivery of glucose to the circulation from antagonizing the essential stress response of glucorticoid in mobilizing peripheral energy stores for emergency purposes.

Immunocytochemical detection of GLUT2 at the rat intestinal brush-border membrane.

We have proposed a new model of intestinal sugar absorption in which high sugar concentrations promote rapid insertion of the facilitative transporter GLUT2 into the brush-border membrane so that absorptive capacity is precisely regulated to match dietary intake during the assimilation of a meal. However, location of GLUT2 at the brush border by immunocytochemistry has been problematical. We report that control of rapid GLUT2 trafficking and the use of an antibody to a sequence within the large extracellular loop of GLUT2 permits localization of GLUT2 at the brush border. To reveal brush-border GLUT2 fully, it is necessary to digest the sugar chain at the glycosylation site close to the antigenic site. In this way, we have demonstrated by immunocytochemistry PKC-dependent changes in the regulation of brush-border GLUT2 in rat jejunum that correspond to those seen by Western blotting. The functional and immunocytochemical data are now reconciled.

Intestinal sugar absorption is regulated by phosphorylation and turnover of protein kinase C betaII mediated by phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent pathways.

Stimulation of intestinal fructose absorption by phorbol 12-myristate 13-acetate (PMA) results from rapid insertion of GLUT2 into the brush-border membrane and correlates with protein kinase C (PKC) betaII activation. We have therefore investigated the role of phosphatidylinositol 3 (PI3)-kinase and mammalian target of rapamycin in the regulation of fructose absorption by PKC betaII phosphorylation. In isolated jejunal loops, stimulation of fructose absorption by PMA was inhibited by preperfusion with wortmannin or rapamycin, which blocked GLUT2 activation and insertion into the brush-border membrane. Antibodies to the last 18 and last 10 residues of the C-terminal region of PKC betaII recognized several species differentially in Western blots. Extensive cleavage of native enzyme (80/78 kDa) to a catalytic domain product of 49 kDa occurred. PMA and sugars provoked turnover and degradation of PKC betaII by dephosphorylation to a 42-kDa species, which was converted to polyubiquitylated species detected at 180 and 250+ kDa. PMA increased the level of the PKC betaII 49-kDa species, which correlates with the GLUT2 level; wortmannin and rapamycin blocked these effects of PMA. Rapamycin and wortmannin inhibited PKC betaII turnover. PI3-kinase, PDK-1, and protein kinase B were present in the brush-border membrane, where their levels were increased by PMA and blocked by the inhibitors. We conclude that GLUT2-mediated fructose absorption is regulated through PI3-kinase and mammalian target of rapamycin-dependent pathways, which control phosphorylation of PKC betaII and its substrate-induced turnover and ubiquitin-dependent degradation. These findings suggest possible mechanisms for short term control of intestinal sugar absorption by insulin and amino acids.

The active and passive components of glucose absorption in rat jejunum under low and high perfusion stress.

In order to determine how perfusion design affects the relationship of the apparent "active" and "passive" components of glucose absorption, rat jejunum was perfused with 50 mM glucose under conditions of low and high mechanical stress. Phloretin or cytochalasin B was used to inhibit GLUT2 and phloridzin to inhibit SGLT1. In low stress perfusions, the ratios of the "passive" to the "active" components determined using phloretin and phloridzin were 2.2 and 0.43, respectively. This discrepancy was explained by the fact that phloridzin inhibits not only SGLT1 but also indirectly that part of the GLUT2-mediated component controlled by SGLT1 through the glucose-induced activation and recruitment of GLUT2 to the brush-border membrane. In high stress perfusions, the ratios of the "passive" to the "active" components determined using phloretin and phloridzin were 0.94 and 0.95, respectively; cytochalasin B gave 0.95. The identity of these results was explained by the observation that the passive component is not dependent on the active component, because glucose-induced activation and recruitment of GLUT2 does not occur in high stress perfusions. Simultaneous inhibition of SGLT1 and GLUT2 in high stress perfusions with phloridzin and cytochalasin B inhibited absorption by 92 +/- 7 %; non-carrier-mediated transport is therefore minimal. Our data provide support for the view that the term "facilitated" should be used to replace the term "passive" in describing the component now known to be mediated by GLUT2. The study of the mechanism and regulation of this facilitated component depends crucially on the design of the perfusion system.