Julie Affleck

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.

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.

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.

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

Glucose absorption in rat jejunum involves Ca2+‐ and PKC βII‐dependent insertion of GLUT2 into the apical membrane. Ca2+‐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 (RLC20), 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 RLC20 phosphorylation in the terminal web and in the levels of apical GLUT2 and PKC βII, but not SGLT1. Perfusion with phloridzin or 75 mm mannitol, removal of luminal Ca2+, or inhibition of unidirectional 45Ca2+ absorption by nifedipine exerted similar effects. ML‐7 had no effect on the absorption of 10 mm Ca2+, nor clearance of [14C]‐mannitol, which was less than 0.7% of the rate of glucose absorption. Water absorption did not correlate with 45Ca2+ absorption or mannitol clearance. We conclude that the Ca2+ necessary for contraction of myosin II in the terminal web enters via an L‐type channel, most likely Cav1.3, and is dependent on SGLT1. Moreover, terminal web RLC20 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 Ca2+ absorption at luminal concentrations prevailing after a meal.

Expression and cellular distribution during development of the peptide transporter (PepT1) in the small intestinal epithelium of the rat.

Abstract. Immunocytochemical distribution in rat small intestine of PepT1, the oligopeptide transporter responsible for nutritionally important peptide uptake from the adult small intestinal lumen, has been measured during development using an antibody to the C-terminal sequence of PepT1. Distribution is exclusively in the apical brush border of enterocytes from both prenatal and mature animals. However, immediately after birth immunolocalisation of PepT1 extends to the subapical cytoplasm and to the basolateral membrane of enterocytes. No staining is found in crypts or over Goblet cells. Our results imply that the peptide transporter at the basolateral membrane in adult rats must be distinct from PepT1. They also shed new light on the trafficking of PepT1 in enterocytes.

Linkage mapping in the oilseed crop Jatropha curcas L. reveals a locus controlling the biosynthesis of phorbol esters which cause seed toxicity

Current efforts to grow the tropical oilseed crop Jatropha curcas L. economically are hampered by the lack of cultivars and the presence of toxic phorbol esters (PE) within the seeds of most provenances. These PE restrict the conversion of seed cake into animal feed, although naturally occurring ‘nontoxic’ provenances exist which produce seed lacking PE. As an important step towards the development of genetically improved varieties of J. curcas, we constructed a linkage map from four F2 mapping populations. The consensus linkage map contains 502 codominant markers, distributed over 11 linkage groups, with a mean marker density of 1.8 cM per unique locus. Analysis of the inheritance of PE biosynthesis indicated that this is a maternally controlled dominant monogenic trait. This maternal control is due to biosynthesis of the PE occurring only within maternal tissues. The trait segregated 3 : 1 within seeds collected from F2 plants, and QTL analysis revealed that a locus on linkage group 8 was responsible for phorbol ester biosynthesis. By taking advantage of the draft genome assemblies of J. curcas and Ricinus communis (castor), a comparative mapping approach was used to develop additional markers to fine map this mutation within 2.3 cM. The linkage map provides a framework for the dissection of agronomic traits in J. curcas, and the development of improved varieties by marker-assisted breeding. The identification of the locus responsible for PE biosynthesis means that it is now possible to rapidly breed new nontoxic varieties.

Identification of a candidate membrane protein for the basolateral peptide transporter of rat small intestine.

A candidate protein for the basolateral peptide transporter of rat jejunum is described. Vascular perfusion of the photoaffinity label, [4-azido-D-phe]-L-ala (2.5mM), had no effect on the transepithelial transport of the non-hydrolysable dipeptide D-phe-L-gln (1mM) from the lumen, its mucosal accumulation or wash-out into the vascular perfusate. When the label was perfused luminally, the transepithelial transport of D-phe-L-gln was inhibited by 38% (P<0.001) and accumulation increased by 62% (P<0.05). These data are consistent with those of a basolateral transporter that is strongly asymmetric in its substrate binding and transport properties. Labelling of basolateral membrane vesicles with [4-azido-3,5-3H-D-phe]-L-ala revealed that the majority of label was incorporated into a single protein of M(r)112+/-2 kDa and pI 6.5. MALDI-TOF analysis of tryptic digests of the protein followed by database searches established that this protein was novel with no obvious similarity to PepT1, the apical membrane transporter.