George L. Kellett

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, α‐gustducin and transducin, to activate phospholipase C β2 and increase intracellular calcium concentration. Intestinal brush cells or solitary chemosensory cells (SCCs) have a structure similar to lingual taste cells and strongly express α‐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 α‐gustducin, transducin or phospholipase C β2 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 ∼ 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 α‐gustducin versus T1R1, transducin and phospholipase C β2. 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.

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.

The facilitated component of intestinal glucose absorption

Over the last decade, a debate has developed about the mechanism of the passive or ‘diffusive’ component of intestinal glucose absorption and, indeed, whether it even exists. Pappenheimer and colleagues have proposed that paracellular solvent drag contributes a passive component, which, at high concentrations of sugars similar to those in the jejunal lumen immediately after a meal, is severalfold greater than the active component mediated by the Na+‐glucose cotransporter SGLT1. On the other hand, Ferraris & Diamond maintain that the kinetics of glucose absorption can be explained solely in terms of SGLT1 and that a passive or paracellular component plays little, if any, part. Recently, we have provided new evidence that the passive component of glucose absorption exists, but is in fact facilitated since it is mediated by the rapid, glucose‐dependent activation and recruitment of the facilitative glucose transporter GLUT2 to the brush‐border membrane; regulation involves a protein kinase C (PKC)‐dependent pathway activated by glucose transport through SGLT1 and also involves mitogen‐activated protein kinase (MAP kinase) signalling pathways. This topical review seeks to highlight the significant points of the debate, to show how our proposals on GLUT2 impact on different aspects of the debate and to look at the regulatory events that are likely to be involved in the short‐term regulation of sugar absorption during the assimilation of a meal.

Simple-sugar meals target GLUT2 at enterocyte apical membranes to improve sugar absorption: a study in GLUT2-null mice

The physiological significance of the presence of GLUT2 at the food‐facing pole of intestinal cells is addressed by a study of fructose absorption in GLUT2‐null and control mice submitted to different sugar diets. Confocal microscopy localization, protein and mRNA abundance, as well as tissue and membrane vesicle uptakes of fructose were assayed. GLUT2 was located in the basolateral membrane of mice fed a meal devoid of sugar or containing complex carbohydrates. In addition, the ingestion of a simple sugar meal promoted the massive recruitment of GLUT2 to the food‐facing membrane. Fructose uptake in brush‐border membrane vesicles from GLUT2‐null mice was half that of wild‐type mice and was similar to the cytochalasin B‐insensitive component, i.e. GLUT5‐mediated uptake. A 5 day consumption of sugar‐rich diets increased fructose uptake fivefold in wild‐type tissue rings when it only doubled in GLUT2‐null tissue. GLUT5 was estimated to contribute to 100 % of total uptake in wild‐type mice fed low‐sugar diets, falling to 60 and 40 % with glucose and fructose diets respectively; the complement was ensured by GLUT2 activity. The results indicate that basal sugar uptake is mediated by the resident food‐facing SGLT1 and GLUT5 transporters, whose mRNA abundances double in long‐term dietary adaptation. We also observe that a large improvement of intestinal absorption is promoted by the transient recruitment of food‐facing GLUT2, induced by the ingestion of a simple‐sugar meal. Thus, GLUT2 and GLUT5 could exert complementary roles in adapting the absorption capacity of the intestine to occasional or repeated loads of dietary sugars.

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/α‐gustducin to activate PLC β2 and PKC βII. We therefore investigated whether non‐sugar nutrients are regulated by taste receptors using perfused rat jejunum in vivo. Under different conditions, we observed a Ca2+‐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 βII, respectively. Addition of l‐glutamate or sucralose to a perfusate containing low glucose (20 mm) each activated PKC βII and decreased apical PepT1 levels and absorption of the hydrolysis‐resistant dipeptide l‐Phe(ΨS)‐l‐Ala (1 mm), while increasing apical GLUT2 and glucose absorption within minutes. Switching perfusion from mannitol to glucose (75 mm) exerted similar effects. l‐Glutamate induced rapid GPCR internalization of T1R1, T1R3 and transducin, whereas sucralose internalized T1R2, T1R3 and α‐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 βII. These data suggest the existence of a wider Ca2+ and taste receptor‐coordinated transport network incorporating other nutrients and/or other stimuli capable of activating PKC βII and additional transporters, such as the aspartate/glutamate transporter, EAAC1, whose level was doubled by l‐glutamate. The network may control energy supply.

Regulation of GLUT5, GLUT2 and intestinal brush-border fructose absorption by the extracellular signal-regulated kinase, p38 mitogen-activated kinase and phosphatidylinositol 3-kinase intracellular signalling pathways: implications for adaptation to diabetes.

We have investigated the role of the extracellular signal-regulated kinase (ERK), p38 and phosphatidylinositol 3-kinase (PI 3-kinase) pathways in the regulation of intestinal fructose transport. Different combinations of anisomycin, PD98059 and wortmannin had very different effects on fructose transport in perfused isolated loops of rat jejunum. Transport was stimulated maximally by anisomycin (2 microM) and blocked by SB203580 (20 microM), confirming involvement of the p38 pathway. PD98059 (50 microM) alone had little effect on fructose transport. However, it had a dramatic effect on stimulation by anisomycin, diminishing the K(a) 50-fold from 1 microM to 20 nM to show that the ERK pathway restrains the p38 pathway. The K(a) for diabetic jejunum was 30 nM and PD98059 had no effect. Transport in the presence of anisomycin was 3.4-fold that for anisomycin plus PD98059 plus wortmannin. Transport was mediated by both GLUT5 and GLUT2. In general, GLUT2 levels increased up to 4-fold within minutes and with only minimal changes in GLUT5 or SGLT1 levels, demonstrating that GLUT2 trafficks by a rapid trafficking pathway distinct from that of GLUT5 and SGLT1. GLUT2 intrinsic activity was regulated over a 9-fold range. It is concluded that there is extensive cross-talk between the ERK, p38 and PI 3-kinase pathways in their control of brush-border fructose transport by modulation of both the levels and intrinsic activities of GLUT5 and GLUT2. The potential of the intracellular signalling pathways to regulate short-term nutrient transport during the assimilation of a meal and longer-term adaptation to diabetes and hyperglycaemia is discussed.

The regulation of GLUT5 and GLUT2 activity in the adaptation of intestinal brush-border fructose transport in diabetes

The adaptation of D-fructose transport in rat jejunum to experimental diabetes has been studied. In vivo and in vitro perfusions of intact jejunum with D-fructose revealed the appearance of a phloretin-sensitive transporter in the brush-border membrane of streptozoto-cin-diabetic rats which was not detectable in normal rats. The nature of the transporters involved was investigated by Western blotting and by D-fructose transport studies using highly purified brush-border and basolateral membrane vesicles. GLUT5, the major transporter in the brush-border membrane of normal rats, was not inhibited by D-glucose or phloretin. In contrast, GLUT2, the major transporter in the basolateral membrane of normal rats, was strongly inhibited by both D-glucose and phloretin. In brush-border membrane vesicles from diabetic rats, GLUT5 levels were significantly enhanced; moreover the presence of GLUT2 was readily detectable and increased markedly as diabetes progressed. The differences in stereospecificity between GLUT2 and GLUT5 were used to show that both transporters contributed to the overall enhancement of D-fructose transport measured in brush-border membrane vesicles and in vitro isolated loops from diabetic rats. However, overall D-fructose uptake in vivo was diminished. The underlying mechanisms and functional consequences are discussed.

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‐β‐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.

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 βII‐dependent mechanism. Since PKC βII requires Ca2+ and glucose is depolarizing, we have investigated whether glucose absorption is regulated by the entry of dietary Ca2+ through Cav1.3 in the apical membrane. When rat jejunum was perfused with 75 mm glucose, Ca2+‐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 45Ca2+ entry by nifedipine and verapamil. At 10 mm luminal Ca2+, 45Ca2+ 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 Cavβ3 in jejunal mucosa; Western blotting and immunocytochemistry localized Cavβ3 to the apical membrane, together with Cav1.3. We conclude that in times of dietary sufficiency Cav1.3 may mediate a significant pathway of glucose‐stimulated Ca2+ entry into the body and that luminal supply of Ca2+ is necessary for GLUT2‐mediated glucose absorption. The integration of glucose and Ca2+ absorption represents a complex nutrient‐sensing system, which allows both absorptive pathways to be regulated rapidly and precisely to match dietary intake.

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.