The mechanism underlying glucose sensing by L cells is a topic of recent debate, with sweet taste receptors (Tas1R2/3) [23
], SGLT1 [12
] and KATP
] each suggested to play a role. We demonstrate here that genetic or pharmacological interference with SGLT1 abolishes glucose-triggered Ca2+
responses and GLP-1 secretion from L cells in primary culture. This is consistent with the inhibition of GLP-1 secretion by phloridzin from perfused intestinal preparations [10
] and the more recent observation of impaired glucose-triggered GLP-1 secretion in Sglt1
knockout mice [14
]. The latter observations are incompatible with the concept of an apically located glucose receptor on the surface of the L cell, as inhibition of glucose absorption would, if anything, tend to increase exposure of such a receptor to luminal glucose. Although they could be explained by a basolaterally expressed receptor which is exposed to elevated glucose concentrations after SGLT1-dependent absorption through enterocytes, the current findings, in which both sides of the cells are exposed to glucose, argue against any major role of an extracellular receptor. This is consistent with our previous observation that artificial sweeteners, at concentrations that saturate Tas1R2/3 receptors, did not trigger GLP-1 release from primary intestinal cultures [7
]. A metabolic sensing mechanism downstream of SGLT1-mediated glucose uptake could also be envisioned. Monitoring intracellular glucose levels, however, allowed us to dissociate the stimulatory action of Na+
-coupled glucose uptake from possible downstream metabolic effects, as SGLT inhibition had only minor effects on intracellular glucose levels, whereas GLUT inhibitors largely abolished glucose uptake in L cells but did not significantly impair GLP-1 secretion. The finding that Sglt1
knockdown in GLUTag cells impaired both glucose- and αMG-triggered secretion argues for a mechanism intrinsic to the enteroendocrine cells, rather than involving coupling through neighbouring enterocytes. It also demonstrates a dominant role of SGLT1 over SGLT3, which is also produced in GLUTag and primary L cells [7
]. We thus conclude that the electrogenic uptake itself via apically localised SGLT1 [7
] is the major glucose-sensing mechanism in L cells.
To enable the use in primary tissues of FRET-based sensors employing YFP and CFP, we generated a new BAC transgenic mouse model expressing the gene encoding Cre
recombinase under control of the proglucagon promoter, targeting enteroendocrine L cells as well as pancreatic alpha cells unlike the shorter promoter constructs used previously to drive Cre
expression in alpha cells [25
]. Quantification by FACS analysis revealed that, although the majority of proglucagon-stained cells in the colon also contained the Cre reporter in GLU-Cre12×tdRFP mice, ~30% did not. This suggests that a small but significant number of L cells may escape Cre recombination, and should be taken into account when GLU-Cre12 mice are used in future conditional gene knockout experiments. Cre-mediated activation of RFP production was also evident in some cells that did not stain for proglucagon, consistent with the observation that a small proportion of the red fluorescent cells in primary colonic cultures did not exhibit morphology typical of L cells. These cells were readily identifiable and could be avoided in single-cell imaging experiments, but are likely to reflect transient transgene activation in a different cell population during development. Similar findings in the other founder GLU-Cre strains (see ESM Table 2
) suggest that this is not merely an artefact of the particular transgene integration site.
Previous analysis detected Glut1
expression in primary murine L cells, with Glut2
evident in L cells from the SI, and Glut1
in those from the colon [7
]. GLUTag cells notably lack Glut2
], but express Glut3
, as determined by Affymetrix microarrays (data not shown). The role of GLUTs in incretin hormone secretion is unclear, as their pharmacological inhibition had no significant effect on glucose-stimulated GLP-1 secretion in primary cultures, although mice lacking GLUT2 showed reduced plasma GLP-1 concentrations following oral glucose, and a lower intestinal GLP-1 content [26
]. Whereas SGLT1 is apically located on L cells [14
], GLUT2 appeared localised to the basolateral surface of L cells and enterocytes, suggesting that intracellular glucose concentrations would reflect basolateral rather than luminal glucose levels. Whereas GLP-1 secretion is predominantly stimulated by oral rather than systemic glucose delivery, GLP-1 release from the perfused pig intestine was found to be influenced also by vascular glucose levels [27
], possibly through alteration of the intracellular glucose concentration in L cells.
Whether intracellular glucose metabolism plays any role in determining GLP-1 secretion remains uncertain. In GLUTag but not primary L cells, we observed a strong inhibition of secretion when glucose uptake was completely blocked and an amplifying action of glucose under depolarising conditions. The balance between the metabolic and electrogenic effects of glucose is thus slightly different between the cell line and primary culture, with a more dominant role for SGLT1-based glucose sensing in the latter. NAD(P)H autofluorescence measurements suggest that GLUTag and primary L cells increase their metabolic rate in response to extracellular glucose elevation, consistent with our previous observation that ATP concentrations in GLUTag cells are elevated upon exposure to 1 mmol/l glucose [28
]. NAD(P)H changes occurring at glucose concentrations above ~1 mmol/l would be consistent with the recruitment of Glucokinase
, which is known to be expressed in enteroendocrine cells [7
]. Glucokinase activity was demonstrable in GLUTag cell extracts, and was responsive to the glucokinase activator, GKA50. The observed S0.5
value (~3 mmol/l) in the absence of GKA50 is lower than the expected value of ~5-8 mmol/l [20
], possibly reflecting incomplete inhibition of hexokinases I–III or additional regulation of enzyme activity by unknown factors in GLUTag cell extracts. GKA50 significantly affected NAD(P)H autofluorescence at 3 mmol/l glucose in GLUTag cells, demonstrating that glucokinase exhibits at least some control over the metabolic flux in L cells, but had only a small effect on glucose-stimulated GLP-1 secretion from GLUTag cells and no effect on secretion from primary cultures.
The present study demonstrates that metabolism plays at best a minor role in glucose-stimulated GLP-1 secretion in primary cultures, consistent with the finding that non-metabolisable glucose analogues such as αMG are effective stimuli of GLP-1 release in vivo and in vitro [10
]. Although phloretin abolished glucose accumulation in GLUTag and primary L cells, its effect on glucose-stimulated GLP-1 secretion was restricted to the cell line, suggesting that glucose metabolism does not enhance secretion in the context of a predominant SGLT1-mediated stimulus. Future work should address whether the glucokinase/KATP
channel machinery exerts longer-term effects on L cells or enables modulation of GLP-1 secretion by neurohormonal or alternative nutritional stimuli.