Philip A. Helliwell

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.

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.

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.

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.

Tripeptide transport in rat lung

Transport of L-alanyl-D-phenylalanyl-L-alanine was investigated with an in situ vascular perfusion preparation of rat lung and brush border membrane vesicles prepared from type II pneumocytes. In the perfused lung 1 mM tripeptide was transported intact from the alveolar lumen to the vascular perfusate at a mean rate of 25.1 +/- 1.29 (3) nmol/min per g dry weight. D-Phenylalanine also appeared in the vascular perfusate at a rate of 21.9 +/- 1.74 (3) nmol/min per g dry weight indicating that 47% of the absorbed tripeptide was split during passage across the epithelial layer. No dipeptide could be detected in the vascular effluent during perfusions with tripeptide. Rapid L-alanyl-D-phenylalanyl-L-alanine uptake occurred with fresh apical membrane vesicles prepared from type II pneumocytes and this was abolished by treatment with 0.1% triton. The related tripeptide, D-alanyl-L-phenylalanyl-D-alanine, was taken up significantly more slowly by the vesicles. D-phenylalanyl-L-alanine and D-phenylalanyl-D-alanine, were also studied with the vascularly perfused preparation; the mixed dipeptide appeared in the vascular perfusate significantly faster than L-alanyl-D-phenylalanyl-L-alanine whereas D-phenylalanyl-D-alanine appeared more slowly and was not hydrolysed.

Evaluation of a Solid-Supported Tagging Strategy for Mass Spectrometric Analysis of Peptides

We have explored two divinylbenzene cross-linked polystyrene supports for use in a solid-supported N-terminal peptide tagging strategy. Resin-bound tags designed to be cleaved in a single step at the N-terminus of peptides have been devised and explored as peptide N-terminal tagging reagents (constructs) for subsequent mass spectrometric analysis. While the brominated tagging approach shows promise, the use of these specific solid supports has drawbacks, in terms of tagging reaction scale, for real applications in proteomics.

Microwave-Assisted Synthesis of Aryl Amide Bonds on Solid Phase

A novel microwave-assisted synthesis of a library of triarylamides has been undertaken on the solid-phase.