Article
Spatiotemporal Modeling of Triggering and Amplifying Pathways in GLP-1 Secreting Intestinal L Cells Alessia Tagliavini1 and Morten Gram Pedersen1,* 1
Department of Information Engineering, University of Padova, Padova, Italy
ABSTRACT Glucagon-like peptide 1 (GLP-1) is secreted by intestinal L-cells, and augments glucose-induced insulin secretion, thus playing an important role in glucose control. The stimulus-secretion pathway in L-cells is still incompletely understood and a topic of debate. It is known that GLP-1 secreting cells can sense glucose to promote electrical activity either by the electrogenic sodium-glucose cotransporter SGLT1, or by closure of ATP-sensitive potassium channels after glucose metabolism. Glucose also has an effect on GLP-1 secretion downstream of electrical activity. An important aspect to take into account is the spatial organization of the cell. Indeed, the glucose transporter GLUT2 is located at the basolateral, vascular side, while SGLT1 is exposed to luminal glucose at the apical side of the cell, suggesting that the two types of transporters play different roles in glucose sensing. Here, we extend our recent model of electrical activity in primary L-cells to include spatiotemporal glucose and Ca2þ dynamics, and GLP-1 secretion. The model confirmed that glucose transportation into the cell through SGLT1 cotransporters can induce Ca2þ influx and release of GLP-1 as a result of electrical activity, while glucose metabolism alone is insufficient to depolarize the cell and evoke GLP-1 secretion in the model, suggesting a crucial role for SGLT1 in triggering GLP-1 release in agreement with experimental studies. We suggest a secondary, but participating, role of GLUT2 and glucose metabolism for GLP-1 secretion via an amplifying pathway that increases the secretion rate at a given Ca2þ level.
INTRODUCTION A variety of specialized cells are involved in the control of body weight and blood glucose levels because of their role in glucose sensing. The impairment of the glucose-sensing machinery may lead to obesity and diabetes. Pancreatic a-cells and b-cells are the typical glucose-sensing cells, and are responsible for secretion of glucagon and insulin, respectively. Furthermore, the gut plays a pivotal role in nutrient absorption and glucose sensing after a meal (1). Indeed, peptides secreted by specialized gut cells, such as intestinal L-cells, have profound and sustained physiological effects on, e.g., appetite and insulin release. L-cells secrete the incretin hormone glucagon-like peptide 1 (GLP-1) in response to food intake. This hormone amplifies insulin secretion, inhibits glucagon secretion, slows gastric emptying, regulates appetite and food intake, and inhibits apoptosis of b-cells (2). Reduction or even complete loss of GLP-1 secretion induces a drop in the incretin effect
Submitted September 29, 2016, and accepted for publication November 29, 2016. *Correspondence:
[email protected] Editor: Richard Bertram. http://dx.doi.org/10.1016/j.bpj.2016.11.3199 Ó 2017 Biophysical Society.
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(2), which has clinical relevance for the development and treatment of type 2 diabetes (3,4). The development of new therapeutic strategies that aim to increase endogenous GLP-1 secretion makes the study of the GLP-1 stimulussecretion pathways of growing interest (1,5). The molecular pathways of the glucose-sensing cells underlying secretion are different. In b-cells glucose enters the cell via the GLUT transporters, inducing glucose metabolism and subsequent closure of ATP-sensitive potassium (KATP) channels, which permits cell depolarization, Ca2þ influx, and insulin release (6). Besides this triggering pathway, insulin secretion is also controlled by amplifying pathways that augment the secretory response at a given level of Ca2þ (7). Early studies with the GLP-1 secreting GLUTag cell line (8) suggested that glucose-sensing in L-cells could involve GLUT2 and K(ATP)-channels as in pancreatic b-cells. However, subsequent studies (9) revealed that another glucose-sensing mechanism is operating in this type of cell. Sodium-glucose cotransporters (SGLTs) link glucose stimulus to electrical activity and hormone secretion by generating a small depolarizing current sufficient to trigger electrical activity, which causes Ca2þ influx and release of
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GLP-1 (9–11). There is evidence that GLUTag and primary L-cells show differences in their electrophysiological properties (12,13), which may underlie the differences in their secretory responses. Indeed, while GLUTag cells seem to use both GLUT2 and SGLT1 to transduce glucose sensing to GLP-1 secretion, primary L-cells exploit mainly SGLT1 (11,14–16). Recently, we investigated the electrophysiological differences between primary L-cells and GLUTag cells using mathematical modeling (17). Our previous model (17) included a careful description of membrane currents, but did not include mechanisms downstream of electrical activity; we particularly did not model Ca2þ levels or secretion. Another key point, which was ignored in our previous model (17), is the spatial location of the two types of glucose transporters in polarized L-cells in situ, where GLUT2 is predominantly located on the basolateral side of L-cells, while SGLT1 is located on the apical side (15). Also, there is good evidence of an amplifying pathway operating in GLUTag cells, because glucose can modulate GLP-1 secretion when Ca2þ is clamped (11), which was not taken into account in our previous work (17). Concerning the spatial organization, to evaluate the subcellular mechanisms underlying the role of SGLT1 sensing in the apical membrane, in contrast to GLUT2 transport at the basolateral membrane, experiments in a physiological setting should be performed, but this is still technically challenging. This kind of experiment would ideally require simultaneous measurements of electrical activity, calcium dynamics, and release from single L-cells without altering the cell polarity. In the absence of experimental data obtained from L-cells in situ, deeper insight into the stimulus-secretion pathways may be achieved by means of a mathematical model. Therefore, a spatiotemporal model that combines electrical activity, glucose transportation, and metabolism as well as GLP-1 secretion and takes into account the spatial configuration of the cell is developed here, starting from our recent model of the electrical activity in primary L-cells (17).
We model active glucose transportation through SGLT1 in the apical side as well as facilitated diffusion via GLUT2 in the basolateral membrane. The subsequent depolarization in response to glucose transportation, diffusion, and metabolism, and the Ca2þ influx due to voltage-dependent Ca2þ channel opening, is simulated and coupled to a simple model of GLP-1 secretion. We validate the model by simulating published experiments conducted in Parker et al. (11) and Kuhre et al. (16) and we find, in compliance with these results, that glucose transportation into the cell through SGLT1 transporters induces Ca2þ influx and release of GLP-1 as a result of membrane depolarization, while glucose metabolism alone is not sufficient to evoke depolarization, resulting instead in lower GLP-1 secretion, suggesting a secondary role of GLUT2 for GLP-1 secretion. The spatiotemporal model is further exploited to investigate the contribution from the glucose-dependent amplifying pathway to GLP-1 secretion. MATERIALS AND METHODS Mathematical spatial modeling Spatiotemporal signaling in L-cells is modeled by a series of partial differential equations. The cell is represented by a cylinder with length L ¼ 28 mm and base radius of 2.8 mm (10,18) (Fig. 1). Because of this cylindrical geometry, the full three-dimensional problem is reduced to a one-dimensional problem by assuming that the concentrations are uniform in tubular cross sections. Thus, the model consists of a series of reaction-diffusion equations,
vci v2 c i ¼ Di 2 þ f ðx; t; zÞ; vt vx
(1)
where ci is a general species; Di is the diffusion coefficient of the species; x is the spatial variable along which the diffusion is considered; and f ðx; t; zÞ is the production-destruction rate per unit of volume for each location x and time t, which may depend on the set of biochemical species z ¼ ðc1 ; c2 ; .ck Þ. The partial differential equations were solved by discretizing the space into a grid with 1 mm between grid points.
FIGURE 1 Schematic representation of the spatial model. Glucose in the lumen is transported into the cell by SGLT1 transporters, while vascular glucose can be transported into the cell by the facilitated-diffusion transporter GLUT2. Voltage-gated Ca2þ channels are located at the basolateral side of the cell along with GLP-1-containing granules ready for fusion. Naþ channels are arranged along the cell length. To see this figure in color, go online.
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Metabolism The reaction-diffusion equation for the glucose concentration ½G is
v½Gðx; tÞ v2 ½Gðx; tÞ ¼ DG þ JGSGLT dðxÞ vt vx2 þ JGGLUT2 dðx LÞ JGutil ðx; tÞ;
(2)
where dðx LÞ is the Dirac delta function centered in x ¼ L and dðxÞ is the Dirac delta function centered in x ¼ 0; JGSGLT is the glucose flux from the lumen into the cell through the sodium/glucose cotransporter SGLT1 placed at the apical side (15); and JGGLUT2 is the glucose flux due to the GLUT2 transporter, located at the basolateral side of the cell. GLUT2 transporter is modeled as a facilitated diffusion transporter, therefore glucose influx and efflux from/into the vein occurs with rate proportional to the concentration gradient. Glucose phosphorylation inside the cell is mainly performed by glucokinase (10,19). The flux-concentration curve of glucokinase is slightly sigmoidal, and glucose utilization JGutil is modeled with a Hill equation (20). We set the diffusion constant DG ¼ 0:6 mm2/ms (21). The spatiotemporal ATP model is given by
v½ATPðx; tÞ v2 ½ATPðx; tÞ ¼ DATP þ JATPprod ðx; tÞ vt vx2 JATPutil ðx; tÞ;
(3)
where DATP ¼ 0.5 mm2/ms (22). When a glucose molecule proceeds through glycolysis and the Krebs cycle, the free energy that is created is used to generate 31 ATP molecules for each glucose molecule consumed (22). Consequently, the ATP production is modeled as JATPprod ¼ 31JGutil , while ATP utilization is described by a Hill equation similarly to glucose utilization. Model details are reported in the Supporting Material.
Electrical activity The electrical activity of primary L-cells is modeled as in Riz and Pedersen (17) with a Hodgkin-Huxley-type model based on patch-clamp data from primary colonic L-cells (13). The model consists of ATP-sensitive K(ATP)-channels, voltage-gated Naþ-, Kþ-, and Ca2þ-channels, and the electrogenic sodium/glucose cotransporter SGLT1. The ATP-sensitive K(ATP)-current is here modeled as
IKðATPÞ ¼ gKðATPÞ OK ðATPÞðV VK Þ;
(4)
with gKðATPÞ denoting the maximal value of the conductance, and OK ðATPÞ denoting the average fraction of open channels depending on the nucleotide concentration (23). Detailed description of the currents is provided in the Supporting Material. Voltage-dependent Ca2þ channels are supposed to be placed on the basolateral side of the cell, where GLP-1 exocytosis occurs (24). Ca2þ enters through the channels and diffuses inside the cell according to
v½Ca2þ ðx; tÞ v2 ½Ca2þ ðx; tÞ ¼ DCa þ fi ðJCa dðx LÞ vt vx2 JSERCA ðx; tÞ JPMCA dðx LÞ þ Jleak ðx; tÞÞ;
(5)
where DCa is the effective diffusion coefficient for Ca2þ, and fi is ratio of free-to-total Ca2þ. The second term in Eq. 5 represents Ca2þ influx,
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JSERCA is the SERCA pump Ca2þ uptake into intracellular stores, and JPMCA is the flux across the cell membrane through the PMCA pump. Detailed descriptions of the fluxes are provided in the Supporting Material. Sodium influx occurs through both SGLT1 transporters and voltagesensitive Naþ channels. We assumed that the channels are distributed along the cell as shown in Fig. 1. This leads to the Naþ reaction-diffusion equation
v½Naþ ðx; tÞ v2 ½Na2þ ðx; tÞ ¼ DNa þ JNa ðx; tÞ vt vx2 þ JNaSGLT dðxÞ JNapump ðx; tÞ;
(6)
where DNa is the diffusion coefficient for Naþ, which is set equal to 0.04 mm2 ms1 (25). Similarly to Ca2þ influx, the second term JNa represents Naþ influx from the Naþ channels; JNaSGLT is the Naþ flux from the lumen associated to the sodium/glucose cotransport; and JNapump models all mechanisms involved in Naþ efflux, e.g., Naþ/Ca2þ and Naþ/Kþ exchangers. Further details are provided in the Supporting Material.
GLP-1 release As most other hormones, GLP-1 is released via Ca2þ regulated exocytosis. As discussed in the Introduction, there is good evidence of glucose-dependent amplification of secretion in GLP-1 secreting cells (11), resembling the well-studied amplifying pathway in pancreatic b-cells (7). Thus, we model the GLP-1 secretion rate SR as a function of simulated Ca2þ and ATP levels at the basolateral side of the cell, where GLP-1 is released into the blood stream, as
kmaxðCaÞ ½Ca2þ SR Ca ; ATP ¼ KmðCaÞ þ ½Ca2þ kmaxðATPÞ ½ATP 1þ ; KmðATPÞ þ ½ATP 2þ
(7)
where kmaxðCaÞ is the maximal exocytosis rate due to local Ca2þ binding to the exocytotic machinery; KmðCaÞ describes the Ca2þ affinity; kmaxðATPÞ is the maximal amplification of the exocytosis rate due to local ATP; and KmðATPÞ is the ATP affinity. All values are given in the Supporting Material.
RESULTS Glucose-sensing mechanism Experiments on primary single L-cells in culture have been conducted in Parker et al. (11). In these experiments the cells were stimulated with glucose, and the effect of SGLT1/GLUT2 transporter inhibition was studied by measuring intracellular glucose concentrations and GLP-1 secretion. Such experiments were conducted with cultures of isolated cells, where the cell polarization is lost. We first use the spatial model to reproduce those experiments but taking into account the cell polarization. To mimic the experimental conditions where glucose stimulus is equally given to the whole cell, we stimulate the model cell at the apical side in the intestinal lumen, where the SGLT1 cotransporters are located, as well as at the basolateral side in the vein where the GLUT2 transporters are placed, with 10 mM of glucose. In the first 5 s of simulation,
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the luminal glucose concentration ð½Gout Þ is 0.03 mM, and the vascular concentration ð½Gext Þ is equal to the intracellular glucose concentration (1 mM), and the cell is in a silent state. The glucose stimulus is given after 5 s (½Gout ¼ 10 mM and ½Gext ¼ 10 mM), and glucose enters the cell from both sides and triggers electrical activity (Fig. 2 A; black bar indicates glucose stimulus). Spatiotemporal simulation results of intracellular glucose dynamics and the average intracellular glucose concentration are shown in Fig. 2, B and C. After exposure to the stimulus, glucose enters the cell from both sides, and the glucose concentration reaches ~9.5 mM after 60 s. Fig. 2 B shows that the glucose uptake rate is higher for the GLUT2 transporter than for the SGLT1 transporter because G increases faster at the basolateral (right) end. The cell depolarization induced by the glucose stimulus leads to opening of voltagegated Ca2þ channels, and consequently to elevation of the intracellular Ca2þ concentration (Fig. 2, D and E). The Ca2þ concentration reaches 4 mM close to the channels
(x ¼ 28 mm) with mean intracellular values of ~1 mM, in agreement with simulation results in pituitary cells at the distance of 1 mm (26). Such Ca2þ concentration elevations stimulate release of GLP-1-containing secretory granules (see below). After a meal, the luminal glucose concentration may reach very high levels and potentially exceed 100 mM. We simulated such a scenario (see Fig. S1 in the Supporting Material), but found no major difference compared to the case reported in Fig. 2. Thus, a glucose gradient across the cell does not modify the overall behavior of our model. SGLT1 triggers electrical activity and Ca2D influx, GLUT2 promotes glucose influx To investigate the role of SGLT1 in glucose entry and GLP-1 secretion, we simulate experiments with the GLUT2 transporter inhibitor phloretin (11,16). Our model results show that the cell depolarizes when stimulated with glucose via A
B
C
D
E
FIGURE 2 Simulation of glucose-sensing mechanisms with default parameters. SGLT1-substrate concentration ½Gout changes from 0.03 mM (no glucose stimulus) to 10 mM and blood glucose levels ½Gext changes from 1 mM (in equilibrium with intracellular glucose concentration) to 10 mM. (Black bar) 10 mM glucose stimulus. (A) Simulation of membrane potential. (B) Intracellular glucose concentration as a function of time and space (color-coded in millimolar). (C) Average intracellular glucose concentration. (D) Intracellular Ca2þ concentration as a function of time and space (color-coded in micromolar). (E) Intracellular Ca2þ concentration at different locations (from upper to lower panel: x ¼ 28, 27, 26 mm). To see this figure in color, go online.
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SGLT1 (Fig. 3 A) with no evident differences compared to the previous simulations (Fig. 2 A). In contrast, when we focus on the intracellular glucose concentration, a conspicuous reduction of the rate of glucose elevation is observed (Fig. 3, B and C). This result, in compliance with experimental results (11), suggests that the major glucose influx is mediated by GLUT2. GLUT2 inhibition also results in lower steady-state glucose concentration (not shown), because the steady-state level is given from the balance between glucose influx and consumption. However, depolarization is not affected by GLUT2 inhibition, implying a marginal role of K(ATP)-channels in triggering electrical activity during glucose stimuli. Cell membrane depolarization opens Ca2þ channels, and Ca2þ enters into the cell in the same amount as when GLUT2 is not inhibited (Fig. 3, D and E). We analyze the contribution of the glucose-sensing mechanism mediated by the GLUT2 by simulating inhibition of
SGLT1 transporters with phloridzin. Experimentally, in primary L-cells, the presence of phloridzin does not substantially affect glucose entry into the cell and elevation in the intracellular glucose concentration is still observed (11). In agreement with this finding, our simulation shows only a slightly reduction in the intracellular glucose elevation (Fig. 4, B and C). On the other hand, with blockage of SGLT1, the associated inward current is no longer generated and the cell is not able to depolarize (Fig. 4 A). Because the intracellular glucose rise is not considerably altered, the model would suggest that glucose metabolism alone is unable to trigger electrical activity. This is because the reduction in K(ATP)-channel conductance is insufficient to allow action potential firing in absence of the depolarizing SGLT1 current. As a consequence of the lack of depolarization, no Ca2þ enters from the voltage-gated Ca2þ channels (Fig. 4, D and E), which is consistent with observations in
A
B
C
D
E
FIGURE 3 Simulation of glucose-sensing mechanisms with GLUT2 inhibition. SGLT1-substrate concentration ½Gout changes from 0.03 mM to 10 mM, and blood glucose concentration ½Gext changes from 1 to 10 mM. (Black bar) 10 mM glucose stimulus; (light-blue bar) simulated GLUT2 blockage with phloretin. (A) Simulation of membrane potential. (B) Intracellular glucose concentration as a function of time and space (color-coded in millimolar). Note the different scale compared to Fig. 2 B. (C) Average intracellular glucose concentration. (D) Intracellular Ca2þ concentration as a function of time and space (color-coded in micromolar). (E) Intracellular Ca2þ concentration at different locations (from upper to lower panel: x ¼ 28, 27, 26 mm). To see this figure in color, go online.
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A
B
C
D
E
FIGURE 4 Simulation of glucose-sensing mechanisms with SGLT1 inhibition. SGLT1-substrate concentration ½Gout changes from 0.03 mM to 10 mM, and blood glucose concentration ½Gext changes from 1 mM to 10 mM. (Black bar) 10 mM glucose stimulus; (orange bar) simulated SGLT1 blockage with phloridzin. (A) Simulation of membrane potential. (B) Intracellular glucose concentration as a function of time and space (color-coded in millimolar). (C) Average intracellular glucose concentration. (D) Intracellular Ca2þ concentration as a function of time and space (color-coded in micromolar). (E) Intracellular Ca2þ concentration at different locations (from upper to lower panel: x ¼ 28, 27, 26 mm). To see this figure in color, go online.
L-cells from sglt1 knockout mice where 10 mM glucose does not trigger a rise in cytosolic Ca2þ levels (11). GLP-1 secretion We now use the Ca2þ and ATP concentrations at the basolateral side of the cell, where granules ready for exocytosis are likely to be located, to drive a simple model of exocytosis to understand the roles of SGLT1 and GLUT2 transporters in GLP-1 secretion. Hence, we first simulate exocytosis in normal conditions, where both glucose transportation mechanisms are operating. We next investigate the effect on GLP-1 exocytosis when one of the two mechanisms is inhibited. A summary of GLP-1 secretion results is shown in Fig. 5. The rate of secretion in each condition is normalized to the response to glucose in the absence of the inhibitors. When the SGLT1 transporter is inhibited, GLP-1 secretion is reduced by ~80% in agreement with experiments (11,15,16), ascribable to the fact that glucose
metabolism alone is not sufficient to trigger cell depolarization and elevate Ca2þ concentrations (Fig. 3). Block of GLUT2 transporters slightly reduces GLP-1 secretion by ~10%, suggesting that cell depolarization is due to the SGLT transporter, though glucose metabolism still plays a minor role in determining GLP-1 secretion. This finding is consistent with experimental results (11,16) where GLP-1 release is induced by nonmetabolizable glucose analogs (e.g., aMG), albeit secretion is slightly lower than with glucose. To investigate the role of K(ATP) channels, we simulated responses to K(ATP)-channel antagonists, such as tolbutamide, which has been shown to trigger electrical activity (27) and GLP-1 secretion (10,16), and to the K(ATP)-channel opener diazoxide, which has been shown to reduce basal (13) and stimulated (16) GLP-1 secretion. In the model, when K(ATP)-channels are closed (by setting the open probability OKðATPÞ to zero), the basal SGLT1 current allows the cell to depolarize sufficiently to activate voltage-gated
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three different conditions (control, SGLT1 inhibition, and GLUT2 inhibition; Fig. 8), to evaluate the contribution of SGLT1/GLUT2 transporters when exocytosis is only glucose-dependent. The model results are in good agreement with the experimental findings (11), confirming a minor amplifying role of glucose metabolism in GLP-1 secretion. DISCUSSION
FIGURE 5 Model results simulating GLP-1 secretion at 10 mM glucose and the effect of phloridzin, a SGLT1 blocker, or phloretin, a GLUT2 inhibitor. Secretion rates are normalized to the secretion response in the absence of inhibitors (Control: 100%).
channels that cause action potential firing (Fig. 6 A). In contrast, simulated diazoxide application ðOKðATPÞ ¼ 0:5Þ interrupts electrical activity caused by 10 mM glucose (Fig. 6 B). These simulated electrophysiological effects result in GLP-1 secretion (Fig. 6 C) being stimulated by tolbutamide and inhibited by diazoxide, even to a point below basal levels, in agreement with experiments (10,13,16). Experimental results in GLUTag cells in Parker et al. (11), where cells were depolarized by KCl and diazoxide, revealed a glucose-dependent contribution affecting GLP-1 secretion. To simulate cell depolarization with external Kþ and the K(ATP)-channel opener diazoxide (11), the model cell is depolarized by setting the Kþ reversal potential VK ¼ 0 mV, and increasing the K(ATP)-channel open probability ðOKðATPÞ ¼ 0:5Þ. The cell is then stimulated with either 0, 1, 5, or 10 mM of glucose (Fig. 7), or as in the previous A
B
The relative roles of SGLT1 and GLUT2 glucose transporters underlying glucose sensing coupled to GLP-1 secretion is a topic of debate. The two possible glucose-sensing pathways inducing electrical activity underlying GLP-1 secretion consist of either an electrogenic SGLT1 transport, or a glucose metabolism that increases intracellular ATP concentrations and closes K(ATP)-channels. The electrophysiological response promotes Ca2þ influx, which triggers exocytosis of hormone-filled granules. Similarly to the situation in pancreatic b-cells (7), we have here presented a model of hierarchical control of GLP-1 secretion from primary L-cells (Fig. 9). Glucose sensing and transport via the electrogenic SGLT1 alone trigger electrical activity and Ca2þ influx (Fig. 3, A, D, and E), which is sufficient to evoke GLP-1 release (Fig. 5), in agreement with experimental studies (10,11,15,16,28). Simulated intracellular glucose and ATP levels are increased much less when glucose enters solely via SGLT1 (Fig. 3, B and C (11)). Thus, the triggering pathway appears to involve electrogenic SGLT1 transport, electrical activity, Ca2þ influx, and exocytosis (Fig. 9). In contrast, when glucose enters via GLUT2 alone, glucose levels increase much more (Fig. 4, B and C), but the calculated ATP concentration and K(ATP)-channel closure are insufficient to induce electrical activity (Fig. 4 A). Again, similar results have been found experimentally (11,15). However, the glucose rise due to influx via GLUT2 has a potentiating role on the exocytotic response to Ca2þ (11), here represented by the amplification due to C
FIGURE 6 Simulation of pharmacological modulations of K(ATP) channel activity. (A) Electrical activity caused by tolbutamide application, simulated by setting the K(ATP) open probability OKðATPÞ ¼ 0 in basal conditions (0.03 mM luminal glucose and 1 mM vascular glucose). (B) Electrical activity interrupted by diazoxide application ðOKðATPÞ ¼ 0:5Þ in the presence of 10 mM luminal and vascular glucose. (C) Normalized simulated GLP-1 secretion rates under basal conditions, or in the presence of tolbutamide (as in A), 10 mM glucose (as in Figs. 2 and 5; 100%), or 10 mM glucose plus diazoxide (as in B).
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A
B
C
FIGURE 7 Exocytosis results simulating cell depolarization and gKðATPÞ opening, at 0, 1, 5, and 10 mM glucose (Gext and Gout ¼ 0, 1, 5, 10 mM). (A) GLP-1 steady-state secretion rates normalized to the response to 10 mM of glucose (100%). (B) Average intracellular glucose concentration and (C) GLP-1 secretion rate as functions of time when stimulated with 0 mM (shaded), 1 mM (dashed), 5 mM (dash-dotted), and 10 mM (solid) glucose at time t ¼ 5 s, as indicated by the black bars.
ATP in the expression for the secretion rate SR. Note that this amplifying pathway is dependent on the triggering pathway: in the absence of electrical activity and Ca2þ influx, secretion is not evoked by the glucose rise. Currently, virtually nothing is known about the mechanisms of the amplifying pathway, and it was here modeled phenomenologically. We used a mathematical spatiotemporal model taking into account the cellular spatial organization to resemble the in vivo configuration. In the isolated intestine, mimicking the in vivo situation, SGLT1 transporters are placed in the apical side of the cell on the luminal side directly exposed to glucose in the intestine (15). It is interesting that vascular glucose dose-dependently can stimulate GLP-1 secretion in the isolated porcine ileum when the lumen is perfused with a perfusate containing 3.5 mM glucose (29). In the hierarchical view proposed here (Fig. 9), the luminal glucose is required and sufficient to
A
stimulate the triggering pathway, whereas vascular glucose entering the L-cells via basolateral GLUT2 operates via the amplifying pathway to augment GLP-1 release. Without luminal glucose, we would expect that vascular glucose is ineffective because the triggering pathway is not operating. This result was indeed found in experiments with isolated rat intestine, where vascular glucose at concentrations of up to 25 mM was without consistent effect on GLP-1 release in the absence of luminal glucose (16). In the same preparation, SGLT1 activation by the nonmetabolizable glucose analog aMG was sufficient to stimulate secretion (via the triggering pathway), but not as efficiently as by glucose (16), which we propose also activates the amplifying pathway (Fig. 9). This amplifying pathway is clearly present in GLUTag cells, but appears not to be operating in cultured, isolated mouse L-cells (11). Future studies should investigate whether this difference is due to species differences
B
C
FIGURE 8 Exocytosis results simulating cell depolarization and gKðATPÞ opening at 10 mM glucose and in the absence or presence of phloridzin (SGLT blocker) and phloretin (GLUT2 blocker). (A) GLP-1 steady-state secretion rates normalized to the response in absence of inhibitors (100%). (B) Average intracellular glucose concentration and (C) GLP-1 secretion rates as functions of time when stimulated at t ¼ 5 s with 10 mM glucose alone (solid), in the presence of phloridzin (shaded), or in the presence of phloretin (dashed). In (C), the shaded curve is virtually coinciding with the solid curve. (Solid bars) Glucose stimulus.
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Tagliavini and Pedersen 6. Ashcroft, F. M., and P. Rorsman. 1989. Electrophysiology of the pancreatic b-cell. Prog. Biophys. Mol. Biol. 54:87–143. 7. Henquin, J. C. 2009. Regulation of insulin secretion: a matter of phase control and amplitude modulation. Diabetologia. 52:739–751. 8. Reimann, F., and F. M. Gribble. 2002. Glucose-sensing in glucagonlike peptide-1-secreting cells. Diabetes. 51:2757–2763. 9. Gribble, F. M., L. Williams, ., F. Reimann. 2003. A novel glucosesensing mechanism contributing to glucagon-like peptide-1 secretion from the GLUTag cell line. Diabetes. 52:1147–1154. FIGURE 9 Hierarchical control of GLP-1 secretion. SGLT1 is responsible for glucose sensing in the triggering pathway (solid arrows) involving electrical activity and Ca2þ elevations, which trigger exocytosis and GLP-1 release. In the amplifying pathway (dashed arrows), GLUT2 promotes glucose influx that increases metabolic factors, e.g., ATP, that augment the secretory response at a given Ca2þ level.
10. Reimann, F., A. M. Habib, ., F. M. Gribble. 2008. Glucose sensing in L cells: a primary cell study. Cell Metab. 8:532–539. 11. Parker, H. E., A. Adriaenssens, ., F. M. Gribble. 2012. Predominant role of active versus facilitative glucose transport for glucagon-like peptide-1 secretion. Diabetologia. 55:2445–2455. 12. Reimann, F., M. Maziarz, ., F. M. Gribble. 2005. Characterization and functional role of voltage gated cation conductances in the glucagonlike peptide-1 secreting GLUTag cell line. J. Physiol. 563:161–175.
or because of the use of isolated cells versus intact intestinal preparations. In summary, we have extended our previous model of electrical activity in L-cells (17) to include spatiotemporal dynamics of Ca2þ, glucose, and ATP, which drive a submodel accounting for triggering and amplification of GLP-1 secretion. Our model explains and reconciles different experimental findings, and suggests that to investigate whether GLP-1 secretion depends on vascular glucose levels in vivo, intestinal glucose should be present to activate the triggering pathway.
13. Rogers, G. J., G. Tolhurst, ., F. Reimann. 2011. Electrical activitytriggered glucagon-like peptide-1 secretion from primary murine L-cells. J. Physiol. 589:1081–1093.
SUPPORTING MATERIAL
18. Petersen, N., F. Reimann, ., E. J. P. de Koning. 2014. Generation of L cells in mouse and human small intestine organoids. Diabetes. 63:410–420.
Supporting Materials and Methods, one figure, and four tables are available at http://www.biophysj.org/biophysj/supplemental/S0006-3495(16) 34268-0.
AUTHOR CONTRIBUTIONS A.T. performed research, prepared the figures, and wrote the article; and M.G.P. designed research, interpreted model results, and wrote the article.
SUPPORTING CITATIONS
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