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Dapagliflozin stimulates glucagon secretion at high glucose: experiments and mathematical simulations of human A-cells
research-article
Author(s):
Morten Gram Pedersen
a
,
1 ,
Ingela Ahlstedt
2 ,
Mickaël F. El Hachmane
2 ,
Sven O. Göpel
b
,
2
Publication date (Electronic):
18 August 2016
Journal:
Scientific Reports
Publisher:
Nature Publishing Group
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Abstract
Glucagon is one of the main regulators of blood glucose levels and dysfunctional stimulus secretion coupling in pancreatic A-cells is believed to be an important factor during development of diabetes. However, regulation of glucagon secretion is poorly understood. Recently it has been shown that Na +/glucose co-transporter (SGLT) inhibitors used for the treatment of diabetes increase glucagon levels in man. Here, we show experimentally that the SGLT2 inhibitor dapagliflozin increases glucagon secretion at high glucose levels both in human and mouse islets, but has little effect at low glucose concentrations. Because glucagon secretion is regulated by electrical activity we developed a mathematical model of A-cell electrical activity based on published data from human A-cells. With operating SGLT2, simulated glucose application leads to cell depolarization and inactivation of the voltage-gated ion channels carrying the action potential, and hence to reduce action potential height. According to our model, inhibition of SGLT2 reduces glucose-induced depolarization via electrical mechanisms. We suggest that blocking SGLTs partly relieves glucose suppression of glucagon secretion by allowing full-scale action potentials to develop. Based on our simulations we propose that SGLT2 is a glucose sensor and actively contributes to regulation of glucagon levels in humans which has clinical implications.
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Most cited references37
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Hypoglycaemia: the limiting factor in the glycaemic management of Type I and Type II diabetes.
P Cryer (2002)
Hypoglycaemia is the limiting factor in the glycaemic management of diabetes. Iatrogenic hypoglycaemia is typically the result of the interplay of insulin excess and compromised glucose counterregulation in Type I (insulin-dependent) diabetes mellitus. Insulin concentrations do not decrease and glucagon and epinephrine concentrations do not increase normally as glucose concentrations decrease. The concept of hypoglycaemia-associated autonomic failure (HAAF) in Type I diabetes posits that recent antecedent iatrogenic hypoglycaemia causes both defective glucose counterregulation (by reducing the epinephrine response in the setting of an absent glucagon response) and hypoglycaemia unawareness (by reducing the autonomic and the resulting neurogenic symptom responses). Perhaps the most compelling support for HAAF is the finding that as little as 2 to 3 weeks of scrupulous avoidance of hypoglycaemia reverses hypoglycaemia unawareness and improves the reduced epinephrine component of defective glucose counterregulation in most affected patients. The mediator and mechanism of HAAF are not known but are under active investigation. The glucagon response to hypoglycaemia is also reduced in patients approaching the insulin deficient end of the spectrum of Type II (non-insulin-dependent) diabetes mellitus, and glycaemic thresholds for autonomic (including epinephrine) and symptomatic responses to hypoglycaemia are shifted to lower plasma glucose concentrations after hypoglycaemia in Type II diabetes. Thus, patients with advanced Type II diabetes are also at risk for HAAF. While it is possible to minimise the risk of hypoglycaemia by reducing risks -- including a 2 to 3 week period of scrupulous avoidance of hypoglycaemia in patients with hypoglycaemia unawareness -- methods that provide glucose-regulated insulin replacement or secretion are needed to eliminate hypoglycaemia and maintain euglycaemia over a lifetime of diabetes.
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Islet beta-cell secretion determines glucagon release from neighbouring alpha-cells.
Homeostasis of blood glucose is maintained by hormone secretion from the pancreatic islets of Langerhans. Glucose stimulates insulin secretion from beta-cells but suppresses the release of glucagon, a hormone that raises blood glucose, from alpha-cells. The mechanism by which nutrients stimulate insulin secretion has been studied extensively: ATP has been identified as the main messenger and the ATP-sensitive potassium channel as an essential transducer in this process. By contrast, much less is known about the mechanisms by which nutrients modulate glucagon secretion. Here we use conventional pancreas perfusion and a transcriptional targeting strategy to analyse cell-type-specific signal transduction and the relationship between islet alpha- and beta-cells. We find that pyruvate, a glycolytic intermediate and principal substrate of mitochondria, stimulates glucagon secretion. Our analyses indicate that, although alpha-cells, like beta-cells, possess the inherent capacity to respond to nutrients, secretion from alpha-cells is normally suppressed by the simultaneous activation of beta-cells. Zinc released from beta-cells may be implicated in this suppression. Our results define the fundamental mechanisms of differential responses to identical stimuli between cells in a microorgan.
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Role of KATP Channels in Glucose-Regulated Glucagon Secretion and Impaired Counterregulation in Type 2 Diabetes
Shu-quan Zhang, Reshma Ramracheya, Carolina Lahmann … (2013)
Introduction Glucagon and insulin are the body’s principal plasma glucose-regulating hormones. They are secreted from the α and β cells of the pancreatic islets, respectively. Physiologically, glucagon is released in response to a fall in plasma glucose levels, an increase in amino acids, and β-adrenergic stimulation (Gromada et al., 2007). Diabetes is a bihormonal disorder involving both inadequate insulin secretion and defective glucagon secretion. The glucagon secretory defects include oversecretion at high glucose (when it is not needed) and inadequate release at low glucose (when it is needed) (Cryer, 2002; Unger and Cherrington, 2012). Whereas the cellular regulation of insulin secretion is fairly well understood (Remedi and Nichols, 2009; Seino et al., 2011), much less is known about the control of glucagon secretion (Gaisano et al., 2012). Hypotheses for the regulation of glucagon secretion include paracrine effects, mediated by factors released from neighboring insulin-secreting β cells or somatostatin-secreting δ cells, or innervation (Gromada et al., 2007). However, in both human and rodent islets, glucagon secretion is strongly inhibited by glucose concentrations that have little stimulatory effect on insulin secretion (Walker et al., 2011), and glucose remains capable of suppressing secretion following pharmacological or immunological inhibition of somatostatin signaling (de Heer et al., 2008; Vieira et al., 2007). Moreover, glucagon secretion responds normally to hypoglycemia after denervation of the pancreas (Sherck et al., 2001). These considerations suggest that α cells, in addition to being under paracrine control, possess an intrinsic glucose-sensing mechanism. This remains poorly defined, but studies on KATP-channel knockout mice indicate that KATP-channels are somehow involved (Cheng-Xue et al., 2013; Gromada et al., 2004; Muñoz et al., 2005; Shiota et al., 2005) and the inhibitory effect of high glucose can be reversed by low concentrations of the KATP-channel activator diazoxide (Göpel et al., 2000b; MacDonald et al., 2007). In pancreatic β cells, closure of these channels by metabolically generated ATP leads to membrane depolarization, electrical activity, and insulin secretion. It is not immediately evident, however, how regulation of the same channels by glucose in α cells could suppress glucagon secretion. To date, most studies have failed to detect an effect of glucose on α cell KATP-channel activity (Barg et al., 2000; Bokvist et al., 1999; Quoix et al., 2009; Ramracheya et al., 2010), but one study reported a small glucose-induced decrease in KATP channel activity that, paradoxically, was associated with stimulation rather than inhibition of glucagon secretion (Olsen et al., 2005). Notably, all these studies of KATP-channel activity used isolated α cells in tissue culture. Tissue culture and/or deprivation of the normal intercellular milieu following islet dissociation may affect α cell function via altered gene transcription, protein expression, or loss of normal paracrine signaling. To avoid such potential confounding effects, the experiments now reported were (whenever technically feasible) performed on α cells in freshly isolated intact islets. Here we show that glucose-induced inhibition of KATP-channels in α cells results in inhibition of glucagon secretion, that the glucagon secretory defects associated with diabetes can be mimicked by experimental conditions leading to a tiny increase in KATP-channel activity, and that glucose-regulated glucagon secretion can be restored in diabetic or metabolically compromised islets by low concentrations of the KATP-channel blocker tolbutamide. Results Glucose Regulates Glucagon Secretion by an Intrinsic, Nonparacrine Mechanism We first established that the inhibitory effect of glucose on glucagon secretion is secondary to glucose metabolism. Mannoheptulose, an inhibitor of glucose phosphorylation, abolished the inhibitory effect of 6 mM glucose on glucagon secretion, without affecting hormone release at 1 mM glucose (Figure 1A). We used the fluorescent probe Perceval to measure the cytosolic ATP/ADP ratio ([ATP]/[ADP]cyt) in α cells of intact mouse islets (Figure 1B). Addition of the mitochondrial uncoupler FCCP promptly decreased [ATP]/[ADP]cyt.. On average, glucose increased [ATP]/[ADP]cyt (measured as relative increase in Perceval fluorescence above basal) by 33% ± 2% (n = 44; p 99%) in α cells. The reported >5-fold greater ATP sensitivity of KATP-channel activity in α cells than in β cells (Leung et al., 2005) might contribute to the strong tonic inhibition of KATP-channels in α cells. Relationship between KATP Channel Activity and Glucagon Secretion Diazoxide activated KATP-channels inhibited by glucose, with half-maximal stimulation occurring at 29 ± 6 μM (n = 5; Figure 3B). Glucagon secretion was measured in the presence of 6 mM glucose (to maximally inhibit secretion) and increasing concentrations of diazoxide. This enables the relationship between KATP conductance (G, determined as above) and glucagon secretion to be determined over a wider range of conductance than can be obtained by varying glucose concentration (Figure 3C). The G-secretion relationship was bell shaped, being maximal at ∼280 pS (3 μM diazoxide), where both G and secretion were similar to that at 1 mM glucose. Importantly, both small (∼100 pS) increases and decreases in G inhibited glucagon secretion. The ability of diazoxide to reverse the effects of glucose cannot be explained by relief from paracrine suppression of glucagon secretion by insulin or somatostatin, as glucose-induced release of these hormones is unaffected by diazoxide concentrations as high as 30 μM and 200 μM, respectively (MacDonald et al., 2007; Zhang et al., 2007). The data of Figure 3C confirm our earlier finding (MacDonald et al., 2007), that low concentrations of diazoxide antagonize the inhibitory effect of glucose on glucagon secretion. We also tested the effects of glucose and diazoxide in the presence of two different amino acid mixtures, AAMa (2 mM) and AAMb (6 mM), designed to simulate the conditions occurring during fasting (Rudman et al., 1989) and following a protein-rich meal, respectively (Figure 3D). AAMa did not detectably enhance glucagon secretion beyond that evoked by 1 mM glucose. Increasing glucose to 6 mM in the presence of AAMa inhibited glucagon secretion as strongly as it did under control conditions, an effect that was prevented by diazoxide. In fact, diazoxide stimulated glucagon secretion in the presence of 6 mM glucose. AAMb stimulated glucagon secretion ∼4-fold at 1 mM glucose and elevation of glucose to 6 mM reduced glucagon secretion by >60%. Again, no inhibitory effect of glucose was observed in the presence of 3 μM diazoxide. These observations argue that KATP-channel closure plays a key role in the modulation of glucagon secretion by glucose in both the absence and presence of amino acids. Membrane Potential-Dependent Reduction of Action Potential Height Figures 2 and 3 suggest KATP-channel closure leads to membrane depolarization and reduced action potential amplitude. We investigated the relationship between interspike membrane potential and the voltage at the peak of the action potential (Figures 4A and 4B). Peak voltage decreased with interspike depolarization, from +18 ± 6 mV at negative membrane potentials (below −60 mV) to −14 ± 2 mV at depolarized potentials (above −40 mV), being half-maximal at −52 ± 3 mV with a slope factor of 4 ± 1 mV (n = 4 cells from four different islets and three different mice; Figure 4C). Using these values, we estimate that the 9 mV depolarization produced by increasing glucose from 1 mM to 6 mM will reduce action potential peak voltage from +6 mV to −9 mV, in reasonable agreement with values observed experimentally (Figure 2A). Thus, the glucose-induced changes in membrane potential are sufficient to account for the reduction of action potential height. Action potential firing in α cells depends on the opening of voltage-gated Na+-channels (Göpel et al., 2000b). We reasoned that glucose-induced membrane depolarization might reduce action potential height by voltage-dependent inactivation of Na+-channels. In agreement with this idea, the Na+-channel blocker TTX (0.1 μg/ml) reduced action potential peak voltage in α cells exposed to 1 mM glucose by 14 ± 4 mV (n = 5; p 4-fold (inset). From the relationship between [K+]o and glucagon secretion we estimate that elevating [K+]o from 3.6 mM to 9 mM is required to produce the same inhibition of glucagon secretion as that resulting from an increase in glucose from 1 mM to 6 mM (arrow). This increase in [K+]o corresponds to an estimated 13 mV depolarization (estimated from the relationship between [K+]o and α cell membrane potential reported previously; De Marinis et al., 2010), which is in reasonable agreement with the 9 mV glucose-induced depolarization observed experimentally (Figure 2A). Glucose Inhibits Glucagon Secretion by Reducing P/Q-Type Ca2+ Channel Activation Figure 5A summarizes the voltage dependence of exocytosis in identified α cells in intact islets. The relationship predicts that the 9 mV reduction in spike height produced by glucose reduces exocytosis by 75%. Glucagon exocytosis is dependent on Ca2+ entry via voltage-gated Ca2+-channels (see Figure S3). The P/Q type Ca2+-channel blocker ω-agatoxin mimicked the inhibitory effect of 6 mM glucose (Figure 5B) and inhibited depolarization-evoked exocytosis (Figure 5C). The reduction in action potential height produced by 6 mM glucose resulted in 66% ± 10% (n = 5; p 13-fold larger in metabolically intact α cells in islets isolated from α-V59M mice than in control islets (∼1.5 nS/pF versus 0.11 nS/pF; p 50% in mutant α cells (Figure 6B), but it still remained >4-fold higher than in control α cells. We next measured α cell electrical activity in intact control and α-V59M islets. In contrast to control cells (which resemble wild-type cells), only 20% (n = 10) of α-V59M α cells were electrically active in 1 mM glucose. The other 80% were inactive and hyperpolarized (−79 ± 3 mV). When glucose was elevated to 15 mM, two cells depolarized and generated electrical activity, five were refractory to glucose but responded to tolbutamide, and one did not respond to either agent (Figure 6C). Glucagon content was similar in wild-type and α-V59M islets, averaging 1,589 ± 213 pg/islet and 2,093 ± 207 pg/islet, respectively. Unexpectedly, although 80% of α-V59M α cells were electrically silent at 1 mM glucose, glucagon secretion from α-V59M islets was not less than from control islets (Figure 6D). Notably, the inhibitory effect of glucose was halved in α-V59M islets (−18% versus −35%) whereas tolbutamide was as inhibitory as in control islets (∼30%). In control islets, adrenaline (5 μM) stimulated glucagon secretion ∼4-fold, but this effect was strongly reduced in α-V59M islets (Figure 6E); the adrenaline-induced stimulation averaged 7 ± 1 and 24 ± 4 pg/islet/hr (p 85% in islets lacking KATP-channels argues that this mechanism is of relatively minor significance. Resting Activity of KATP Channels Determines Islet Hormone Release The model outlined above postulates that the KATP-channel plays a dual role in the regulation of insulin and glucagon secretion. Depending on the initial activity, channel closure may either stimulate (β cells) or inhibit (α cells) secretion. Thus, increasing basal KATP activity in α cells may result in a β cell phenotype—i.e., no electrical activity or glucagon secretion at 1 mM glucose, and stimulation of both by glucose. This is precisely what is seen in the presence of low concentrations of diazoxide (Figure 7B). Similarly, we observed that glucose stimulates electrical activity in some α-V59M α cells (Figure 6C). Figure S6 illustrates the relationship between KATP-channel activity and glucagon secretion. Cells with little or no expression of mutant channels behave like control cells (Figure S6A). Cells expressing high levels of mutant channels (or exposed to high concentrations of diazoxide) will be permanently hyperpolarized and show no glucagon secretion at either 1 or 6 mM glucose (Figure S6B). Cells lying between these extremes will show a spectrum of responses: in some, 6 mM glucose will stimulate glucagon secretion (Figure S6C); in others, glucagon release will be high and unaffected by glucose (Figure S6D). It is of interest that the whole-cell KATP conductance is ∼3-fold larger in isolated rat α cells (Olsen et al., 2005) than that we observe in intact mouse islets. This may account for the paradoxical glucose-induced stimulation of glucagon secretion observed in single α cells. Recently it was reported that the impaired counterregulation of glucagon secretion in diabetic rats could be restored by a somatostatin receptor antagonist (Yue et al., 2012), suggesting that somatostatin signaling is enhanced in diabetic islets. The effects of somatostatin on α cells include activation of K+-channels (GIRK) (Kailey et al., 2013). The resulting increase in GIRK-channel activity could be envisaged to have the same effect on glucagon secretion as a low concentration of diazoxide or weak expression of mutant KATP-channels. Unexpectedly, glucagon secretion is close to normal in α-V59M islets, although α cell electrical activity is strongly affected (Figures 6C and 6D). This is consistent with the mild metabolic phenotype of α-V59M mice (Figures S4A and S4B) and suggests that glucagon secretion is upregulated in the 20% of α cells that remain active. However, the response to adrenaline was reduced by >70% (Figure 6E) in α-V59M islets, a finding that may explain the impaired insulin tolerance of α-V59M mice (Figure S4C). The stimulatory effect of adrenaline on glucagon secretion involves electrical activity and requires influx of extracellular Ca2+ (De Marinis et al., 2010). Thus, it seems likely that in α cells expressing mutant KATP-channels, which are electrically silent, adrenaline will be without stimulatory effect. Collectively, these findings argue that the subset of α cells in α-V59M islets that remain electrically active operate close to their maximum secretory capacity already at 1 mM glucose, accounting for the smallness of the adrenaline effect. This is reminiscent of reports of near-normal glucagon secretion in islets after almost total (98%) ablation of α cells by diphtheria toxin (Thorel et al., 2011). If this hypothesis is correct, then glucagon secretion evoked by experimental paradigms that bypass action potential firing should be enhanced in α-V59M islets. Indeed, high-[K+]o depolarization (which stimulates secretion in both electrically active and electrically silent α cells) produces a much larger stimulation of glucagon secretion in α-V59M than in control islets (Figure 6F). It may seem surprising that glucose remains capable of inhibiting glucagon secretion in α-V59M islets despite no detectable glucose-induced reduction of KATP-channel activity. However, for the reasons we outlined above, glucagon secretion will principally reflect the activity of the small subset of cells in which KATP-channel activity is normal. By contrast, measurements of KATP-channel activity will be dominated by α cells expressing high levels of mutant KATP channels, which respond poorly to glucose. Importantly, in some α cells, expression of mutant KATP-channels is low and total KATP-channel activity only marginally increased, just sufficient to suppress electrical activity at 1 mM glucose. However, when glucose is elevated and KATP-channel activity is reduced, these α cells undergo depolarization and start firing action potential with resultant stimulation of glucagon secretion. This opposes the glucose-induced inhibition in other cells and explains the reduced glucose-induced inhibition of glucagon secretion in α-V59M mutant islets. In general, the subtle impact of targeting the Kir6.2-V59M mutation to the α cell on glucagon secretion suggests that enhanced α cell KATP-channel activity is unlikely to affect glucagon secretion in patients with neonatal diabetes due to gain-of-function KATP-channel mutations. Implications for Type 2 Diabetes In T2D, glucagon secretion in vivo is often stimulated rather than inhibited during hyperglycemia (Dunning et al., 2005). We observed a similar abnormality in 50% of islet preparations from T2D organ donors (Figure 7A); the remaining preparations exhibited normal glucose regulation. Thus, T2D islets are heterogeneous with respect to the dysregulation of glucagon secretion, in agreement with a recent report (Li et al., 2013). Intriguingly, the inverted response to glucose seen in some T2D islet preparations can be mimicked in ND islets by a small increase (0.5%) in KATP-channel activity produced by diazoxide (Figure 7B). Similarly, metabolic inhibition, which activates KATP-channels, affects glucagon secretion in the same way as T2D (Figure 7D). This suggests that T2D may be associated with increased KATP-channel activity, possibly as a consequence of impaired islet metabolism (Doliba et al., 2012). It is notable that glucagon secretion in the presence of diazoxide or oligomycin shows not only an inverted glucose response but also reduced secretion at 1 mM glucose. This is reminiscent of the impaired counterregulation that is a feature of longstanding T2D, raising the interesting possibility that T2D may involve a progressive and time-dependent increase in K+-channel activity and deterioration of the metabolic regulation of glucagon secretion. It is therefore of interest that a low concentration of the KATP-channel blocker tolbutamide restores normal glucose regulation of glucagon secretion in metabolically compromised islets (Figure 7D) and improves it in islets from donors with T2D (Figures 7E and 7F). Possibly, low-dose sulfonylurea (much less than required to stimulate insulin secretion) may be a useful addition to insulin therapy. Finally, our data suggest that a single cellular disturbance (impaired glucose metabolism and ATP production), via increased KATP-channel activity, may explain the trio of hormone secretion defects associated with T2D: impaired glucose-induced insulin secretion, inverted glucose regulation of glucagon secretion, and defective counterregulation. Experimental Procedures All experiments were conducted in accordance with the UK Animals Scientific Procedures Act (1986) and University of Oxford ethical guidelines. Media Media used are specified in Table S1. Animals and Generation of α-V59M Mice Most experiments were performed on islets isolated from NMRI mice obtained from a commercial supplier. Mice expressing Kir6.2-V59M in α cells (α-V59M mice) were generated using a Cre-lox approach (Clark et al., 2010). Human Islets Human islets (obtained with ethical approval and clinical consent) were isolated from pancreases of 46 nondiabetic donors and 10 donors with T2D. Genotyping of Human Islet DNA The rs5219 variant was genotyped using an allelic discrimination assay-by-design method on an ABI 7900 analyzer (Applied Biosystems). Hormone Release Measurements Measurements of insulin and glucagon secretion were performed using the in situ pancreas perfusion or static incubations of isolated islets. Electrophysiology All electrophysiological measurements were performed at +34°C on α cells within intact islets (from NMRI or α-V59M and control mice). For the membrane potential and whole-cell KATP-current recordings (Figures 2A–2C), the perforated patch technique was employed as reported previously (De Marinis et al., 2010). The pipette solution consisted of IC1, and the bath contained EC2. Exocytosis was measured as increases in membrane capacitance in α cells in intact islets as described previously (Göpel et al., 2004) using pipette medium IC2 and extracellular medium EC3. The impact of glucose on exocytosis was tested using the perforated patch technique using pipette medium IC3. Voltage-dependent inactivation of the Na+-current was evaluated using the standard whole-cell technique and a two-pulse protocol using intra- and extracellular media IC4 and EC4, respectively. ATP Imaging The ATP/ADP sensor Perceval (Berg et al., 2009) was used as previously described (Tarasov et al., 2012). Identification of α Cells The identity of the α cells was established either by (1) immunocytochemistry following injection of the cell with biocytin (0.5 mg/ml) via the recording electrode (Figures S2C and S2D) or (2), in the case of α-V59M and control α cells, by tdRFP fluorescence. In perforated patch measurements of electrical activity and KATP-channel activity in NMRI islets (Figures 2–4), it was not always possible to identify the cell by immunocytochemistry because the cell detached when retracting the recording electrode. In these cases, α cells were identified by their spontaneous action potential firing at 1 mM glucose (Göpel et al., 2000a). Statistical Analysis Details on the analysis of the electrophysiological data are given in the Supplemental Information. Data are presented as man values ± SEM of the indicated number of experiments (n). Error bars in figures represent SEM. Statistical significances were, unless otherwise indicated, evaluated using Student’s t test.
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Journal
Journal ID (nlm-ta): Sci Rep
Journal ID (iso-abbrev): Sci Rep
Title:
Scientific Reports
Publisher:
Nature Publishing Group
ISSN
(Electronic):
2045-2322
Publication date
(Electronic):
18
August
2016
Publication date Collection: 2016
Volume: 6
Electronic Location Identifier: 31214
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Publisher Item ID: srep31214DOI: 10.1038/srep31214
PMC ID: 4989223
PubMed ID: 27535321
SO-VID: cb250c1b-bb53-4501-8d95-1cbcd79be3fe
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This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
History
Date
received
: 12
January
2016
Date
accepted
: 07
June
2016
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