Regulation of glucagon secretion by glucose: paracrine,intrinsic or both?J. N. Walker1,2, R. Ramracheya1, Q. Zhang1,3, P. R. V. Johnson2, M. Braun1,3 & P. Rorsman1,3
1Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford OX3 7LJ, UK2Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, UK3Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada T6G 2E1
Glucagon secretion is regulated by glucose but the mechanisms involved remain hotly debated. Both intrinsic (within the α-cell itself) andparacrine (mediated by factors released β- and/or δ-cells) have been postulated. Glucagon secretion is maximally suppressed by glucoseconcentrations that do not affect insulin and somatostatin secretion, a finding that highlights the significance of intrinsic regulation of glucagonsecretion. Experiments on islets from mice lacking functional ATP-sensitive potassium channels (KATP-channels) indicate that these channels arecritical to the α-cell’s capacity to sense changes in extracellular glucose. Here, we review recent data on the intrinsic and paracrine regulationof glucagon secretion in human pancreatic islets. We propose that glucose-induced closure of the KATP-channels, via membrane depolarization,culminates in reduced electrical activity and glucagon secretion by voltage-dependent inactivation of the ion channels involved in actionpotential firing. We further demonstrate that glucagon secretion measured in islets isolated from donors with type-2 diabetes is reduced at lowglucose and that glucose stimulates rather than inhibits secretion in these islets. We finally discuss the relative significance of paracrine andintrinsic regulation in the fed and fasted states and propose a unifying model for the regulation of glucagon secretion that incorporates bothmodes of control.Keywords: glucagon, insulin, KATP-channels, paracrine, type-2 diabetes, α-cell, β-cell
Date submitted 1 April 2011; date of final acceptance 4 May 2011
IntroductionThe pancreatic islets play a central role in systemic fuelhomeostasis. They do so by secreting several hormones thatdirectly influence important metabolic pathways that regulateuptake, deposition and usage of fuels. The two most importanthormones in this context are insulin (released by the β-cells)and glucagon (secreted by the α-cells). Insulin and glucagon aresecreted reciprocally in response to fluctuations in the plasmaglucose levels (and other metabolic fuels). In the fed state,insulin predominates and this favours the uptake of glucoseand lipids in skeletal muscle, liver and fat cells. In the fastedstate, insulin secretion is reduced and glucagon secretion isenhanced, leading to stimulation of catabolic processes and themobilization of glucose and free fatty acids [1]. The concertedactions of insulin and glucagon ensure that plasma glucoselevels are normally kept within a relatively narrow range.
It is now recognized that pancreatic islet dysfunction isa hallmark of both human type-1 and type-2 diabetes [2].This is manifested as insufficient release of insulin combinedwith impaired regulation of glucagon secretion [3]. Theabnormalities of glucagon secretion are twofold: too muchglucagon is secreted during hyperglycaemia and too little isreleased during hypoglycaemia [4].
Correspondence to: Patrik Rorsman, Oxford Centre for Diabetes, Endocrinology andMetabolism, University of Oxford, Churchill Hospital, Oxford OX3 7LJ, UK.E-mail: [email protected]
The relative contributions of insulin deficiency andglucagon excess to the hyperglycaemia of diabetes remaindebated. However, the recent experiments using glucagonreceptor knockout mice (GlcR−/−) in conjunction withstreptozotocin-induced destruction of the β-cells indicate thathyperglucagonaemia may be far more important than perhapspreviously recognized [5]. These observations underscore therole of glucagon in diabetes and indicate that pharmacologicalmodulation of glucagon release may represent a means toachieve improved glycaemic control in diabetes. Indeed, theintroduction of therapies based on glucagon-like peptide-1(GLP-1), which affects both insulin and glucagon secretion [6],illustrates the potential of the α-cells as a pharmacologicaltarget.
During the last 25 years, there have been significant advancesin several areas pertinent to the understanding of insulinsecretion [7]. Key components of the β-cell stimulus-secretioncoupling include glucokinase (which is rate-limiting for glucosemetabolism), the ATP-sensitive potassium channels (that linkmetabolically induced changes in cytoplasmic ATP/ADP-ratioto changes in β-cell membrane potential) and voltage-gatedCa2+-channels (that mediate the Ca2+-entry that triggersexocytosis of the insulin-containing secretory granules). Insulinis also released in response to elevation of other nutrients suchas amino acids [8] and free fatty acids [9] by mechanisms thatinvolve (but are not restricted to) stimulation of β-cell electricalactivity.
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Much less is known about the regulation of glucagon secre-tion. In rodents, α-cells comprise only 10–15% of the islet cellpopulation. Whereas intact islets may thus be regarded as anacceptable approximation of the β-cell (75–80% of the isletcells in mice), this clearly does not apply to the α-cells. Eluci-dation of the properties and regulation of the α-cell thereforerequire methods that allow the α-cell to be studied in isola-tion. Electrophysiological studies on individual α-cells (eithersingle α-cells in cell culture or α-cells within intact islets) haverevealed that they are electrically excitable and that stimulationof glucagon secretion is secondary to increased action poten-tial firing and elevation of cytoplasmic [Ca2+]i [10]. Becauseglucagon secretion is exaggerated in diabetes, an effect which is,at least partially, corrected by insulin therapy, it has been pro-posed that glucagon secretion is normally under paracrine con-trol by insulin [11] or other factors like GABA [12] or Zn2+ [13]secreted from neighbouring β-cells. In addition, somatostatin(released from the pancreatic δ-cell) could function as aparacrine inhibitor of glucagon secretion [14,15]. The con-cept that glucagon secretion is regulated by paracrine processesis supported by reports that isolated α-cells (in which intra-isletinsulin signalling is physically disrupted) respond inappropri-ately to elevation of glucose with stimulation of glucagonsecretion [16,17]. However, other studies indicate that singleα-cells remain capable of responding to glucose with loweringof [Ca2+]i [18] and inhibition of glucagon secretion [15]. Thus,it seems likely that the α-cell is not only under paracrine con-trol but also equipped with an intrinsic regulation. The natureof this intrinsic regulation remains poorly defined but thereis evidence for the involvement of ATP-sensitive potassiumchannels (KATP-channels) of the same type as those found inβ-cells [19–21]. Another important question is whether dataobtained in rodent islets can be extended to human islets.Recent work indicates that the electrophysiological propertiesof rodent and human α-cells differ with regard both to ionchannel complements expressed and the biophysical propertiesof these channels [22,23]. With regard to the paracrine control,it is also worthy of note that whereas rodent α-cells containGABAA receptor Cl−-channels that influence glucagon secre-tion [12,24,25], such channels are present at very low density(if at all) in human α-cells [26]. Thus, paracrine/intercellularsignalling might be quite different in rodent and human islets.
Here, we first evaluate the relative importance of intrinsic andparacrine mechanisms in the regulation of glucagon secretionby glucose in human pancreatic islets. We then considera possible mechanism for intrinsic regulation of glucagonsecretion and how it may become disrupted in type-2 diabetes.We finally propose a unifying model that incorporates bothmodes of the regulation of glucagon secretion.
Effects of Glucose on Pancreatic HormoneSecretionThe notion that the α-cell might be under paracrine control bya product released by the β-cell goes back to 1969 when it wasreported that the lack of insulin in diabetes is associated withoversecretion of glucagon (review: [27]). Paracrine regulationof the α-cell is facilitated by the architecture of the islet
with the α-cells in close proximity to the β-cell, especiallyin human islets [28,29]. Clearly, a prerequisite for paracrineregulation is that the paracrine regulator is present under thephysiological conditions where it is supposed to act. How wellis this requirement fulfiled by factors released from the β- orδ-cells? We reasoned that if glucose controls glucagon secretionby a paracrine effect mediated by products released from the β-and δ-cells, then inhibition of glucagon secretion should exhibitthe same glucose dependence as the stimulation of insulinand somatostatin secretion. Our analyses indicate that thiscondition is not fulfiled by insulin and somatostatin. Figure 1Aand B summarizes the concentration dependence of the effectsof glucose on pancreatic hormone secretion from mouse isletsand human islets, respectively. In human and mouse isletsalike, glucagon secretion is nearly maximally inhibited by3 mM glucose, a concentration not associated with any majorstimulation of insulin or somatostatin secretion. In additionto the inhibitory effect exerted at glucose concentrations<6 mM, glucose also exerts a paradoxical ‘stimulatory’ effectat higher glucose concentrations. Thus, 20 mM glucose issignificantly less inhibitory than 5 mM (mouse islets; [30]) and6 mM (human islets; [26]). The underlying mechanism hasnot been resolved but it may be relevant (for reasons that willbecome clear below) that it occurs over the range of glucoseconcentrations associated with stimulation of insulin secretion.
It could be argued that glucagon is regulated by such minuteand local changes in the intra-islet somatostatin levels thatthey escape detection in static incubations of whole islets. Wetherefore tested the effects of glucose (6 mM) on glucagonsecretion from human islets in the absence and presence ofthe somatostatin receptor 2 (SSTR2), the receptor subtypepresent in human α-cells, antagonist CYN-154806 (figure 2A).However, glucose retained an inhibitory effect in the presenceof this inhibitor.
We also tested the effects of exogenous insulin; althoughinsulin itself exerted a strong inhibitory effect on glucagonsecretion when applied at 1 mM glucose (i.e. under conditionswhen insulin is not secreted), it stimulated glucagon secretionfrom human islets when tested in the presence of 6 mMglucose (figure 2B). We acknowledge that the concentrationused (100 nM) is high compared to circulating insulinlevels (increase from basal 30 to 300 pM following a mixedmeal; [31]). However, the intra-islet concentration is likely tobe at least as high. The average intra-granular concentration ofinsulin is approximately 100 mM [7]. Thus, the granule contentcan be diluted 1 million times and yet insulin will be presentat concentrations comparable to those used here. It shouldbe recognized, however, that the response to a permanentelevation of insulin produced by application of exogenous maybe quite different from that occurring in intact islets duringrelease of endogenous insulin, which results in brief pulses (<1to 60 s) of insulin [32].
We have previously reported that addition of Zn2+ (30 μM),co-released with insulin when the zinc-insulin crystal withinthe granule lumen is dissolved, unlike what has been observedin rodent islets [13], stimulates rather than inhibits glucagonsecretion from human islets.
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Figure 1. Glucose dependence of pancreatic hormone secretion from mouse (A) and human (B) pancreatic islets. Data for insulin, glucagon andsomatostatin have been measured from the same islets at 0–20 (mouse) and 1–20 mM glucose (man). Data are mean values ± s.e.m. of 8 (A) and 7–12(B) experiments. In (B), the individual experiments were conducted on islets from 3 to 4 different pancreases. Shaded area indicates range of glucoseconcentrations over which most of the regulation of the inhibition of glucagon secretion occurs. All values are statistically different (p < 0.05, or better)for all values measured at 3 mM glucose and above except for insulin secretion in mouse islets where the difference first becomes statistically different at10 mM. †p < 0.05 (or better) for glucagon secretion at 20 mM glucose versus that at 5 mM (mouse) or 6 mM (human).
Collectively, the experiments summarized in figures 1 and 2,indicate that the effects of glucose on glucagon secretion, atleast for the responses to concentrations up to 6 mM, are notmediated by paracrine effects mediated by factors released fromβ-cells (insulin, Zn2+ or GABA) or δ-cells (somatostatin).In fact, stimulation of insulin secretion might underlie theparadoxical increase in glucagon secretion observed whenglucose is elevated above 6 mM.
Adrenaline Does Not Stimulate GlucagonSecretion From Human IsletsIn rodent islets, adrenaline (1–5 μM) is a powerful stimulusof glucagon secretion [15,33]. This effect is mediated by an
elevation of cAMP and is mimicked by high concentrationsof the adenylate cyclase activator forskolin [33]. Surprisingly,adrenaline was without stimulatory effect on glucagon secretionfrom human islets (figure 3A). It could be argued that the stim-ulatory effect of adrenaline is obscured by simultaneous activa-tion of inhibitory α2-adrenoreceptors (cf. [34]). However, thispossibility seems less likely given that the adenylate cyclase acti-vator forskolin (even when applied at a concentration of 5 μM)likewise failed to enhance glucagon secretion (figure 3B).
We also considered the possibility that the simultaneousstimulation of somatostatin inhibits glucagon secretion butbelieve it can be discarded because adrenaline was without effecton glucagon secretion in the presence of the SSTR2 antagonistCYN154806 (not shown). The inhibitory effects of somatostatin
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Figure 2. Effects of insulin and somatostatin on glucagon secretion inhuman islets. Glucagon secretion measured at 1 and 6 mM glucose ([Glc])in the absence and presence of 100 nM of the somatostatin receptorsubtype 2 antagonist CYN154806 (A) and 100 nM insulin (B) as indicated.Data are mean values ± s.e.m. of 10–12 (A) and 9–14 (B) experimentswith islets from four different donors for each set of experiments. *p < 0.05and **p < 0.01 for the effects versus secretion at 1 mM glucose. †p < 0.05and ††p < 0.01 for effect insulin versus corresponding condition in theabsence of the respective compound.
Figure 3. Adrenaline and forskolin are weak stimuli of glucagon secretionin human islets. Glucagon secretion measured at 1 mM glucose in theabsence and presence of 5 μM adrenaline (A) and 10 μM forskolin (B).Data are mean values ± s.e.m. of 32–37 experiments with islets from11 donors (A) and 9–13 experiments with islets from 3 donors (B).
on exocytosis/hormone secretion in rodent α- and β-cells aremediated by activation of pertussis toxin-sensitive inhibitoryG-proteins (Gi) [34,35]. Surprisingly, pretreatment of human
Figure 4. Pertussis toxin acts as an ‘islet activating protein’. Glucagon (A),insulin (B) and somatostatin (C) secretion measured from humanpancreatic islets at 1 and 6 mM glucose ([Glc]) without (black bars)and with (grey bars) pretreatment for >4 h with 100 ng/ml pertussistoxin. Data are mean values ± s.e.m. of 10–11 experiments with islets fromthree different donors. *p < 0.05 (or better) for the effects versus secretionat 1 mM glucose. †p < 0.05 (or better) for effect of pertussis toxin versuscorresponding condition in the absence of the toxin.
islets with pertussis toxin inhibited glucagon secretion at 1 mMglucose as strongly as 6 mM glucose in untreated islets andglucose exerted no additional inhibitory effect in toxin-treatedislets (figure 4A). The suppression of glucagon secretion wasparalleled by a greater than fourfold stimulation of insulinsecretion at both 1 and 6 mM glucose (figure 4B). Somatostatinwas also stimulated by pretreatment with pertussis toxin; releaseat 1 and 6 mM glucose was enhanced greater than threefold
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DIABETES, OBESITY AND METABOLISM review articleand approximately 12-fold, respectively (figure 4C). Thus, atleast in human islets and in terms of its effect on insulinand somatostatin secretion, pertussis toxin certainly lives upto its old name ‘islet-activating protein’ [36]. The stimulationof somatostatin secretion (although formidable) is unlikely toexplain the suppression of glucagon secretion at 1 mM glucoseas any such effects should have been prevented by pretreatmentwith the toxin [34]. Rather, we attribute the effects on glucagonrelease to the enhancement of insulin secretion.
The poor stimulatory effect of adrenaline on glucagonsecretion from human islets may explain why hypersecretionof insulin due to loss-of-function mutations in the KATP-channel results in more severe hypoglycaemia in man [37]than in mice [38]. Possibly, mice cope better with insulin-induced hypoglycaemia than humans because of a strongeradrenaline-induced counter-regulatory stimulation of their α-cells. There is some evidence that the weak effect on glucagonsecretion is echoed by poor sympathetic innervations of humanα-cells [39]. It is unlikely that the weak stimulatory effects ofadrenaline on glucagon secretion can be accounted for by poorislet quality. The same negative observations were consistentlymade with islets from a total of 11 different, which invariablyresponded to glucose with stimulation (insulin or somatostatin)or inhibition (glucagon) secretion [23,26].
Role of KATP-Channels in Glucagon SecretionKATP-channels play a key role in the regulation of insulinsecretion by glucose in rodents [40]. Human α-cells alsocontain KATP-channels [23] and application of the KATP-channel blocker tolbutamide produces strong depolarization(figure 5A). This finding argues that KATP-channels are activein the α-cell and that they exert a repolarizing function. Impor-tantly, KATP-channel activity in metabolically intact α-cells isalmost maximally inhibited even in the absence of glucose andaddition of tolbutamide produces no detectable further inhi-bition. Thus, even very small changes in KATP-channel activityhave dramatic effects on the membrane potential and electricalactivity of the human α-cell. In addition to depolarizing theα-cell (and sometimes increasing action potential frequency),tolbutamide consistently and dramatically reduced the peakvoltage of the action potentials (figure 5B). In human α-cells,action potentials recorded in the presence of 1 mM glucoseoften overshoot and peak at +20 mV but stop at −20 mV inthe presence of the KATP-channel blocker. Similar effects ofglucose and tolbutamide have been reported in isolated ratα-cells (see Fig. 2 in [17]).
The observed decrease in action potential amplitude weattribute to voltage-dependent inactivation of the ion chan-nels involved in action potential firing. Many ion channels
Figure 5. Voltage dependence of exocytosis in human α-cells. (A) Electrical activity recorded from an α-cell in an intact human pancreatic islet exposedto 1 mM glucose and following addition of 400 μM tolbutamide. Note that the α-cell is firing action potentials at 1 mM glucose and that applicationof tolbutamide depolarizes the α-cell, increases action potential frequency and lowers the action potential amplitude. (B) Examples of action potentialsrecorded in the absence (•1 ) and presence of tolbutamide (•2 ), taken as indicated in A. The horizontal dashed line indicates that action potentials in thepresence of tolbutamide peak at −20 mV. (C) Voltage dependence of α-cell exocytosis. All responses have been normalized to the maximum exocytoticresponse (usually at 0 or +10 mV).
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(including those involved in α-cell action potential generation)exhibit a dual dependence on voltage [41]. Short depolariza-tions increase channel activity, whereas protracted membranedepolarizations make the channels enter a non-conductinginactivated state. Reactivation of inactivated ion channelsrequires brief (∼100 ms) exposure to a negative membranepotential. It is thus possible that the membrane depolarizationresulting from closure of the KATP-channels prevents reactiva-tion of the channels and that this accounts for the observedreduction of action potential height (which depends on themagnitude of the currents involved in action potential firing).
Why do we emphasise the effect of tolbutamide onthe peak voltage of the action potential? Pancreatic α-cellsdiffer from β-cells in being electrically active at low glu-cose [12,16,17,22,42,43]. In β-cells, membrane depolarizationand induction of action potential firing will increase insulinsecretion from a very low basal level [7]. By contrast, in α-cellssecretion is ongoing at low glucose and although a glucose-induced depolarization as such might increase action potentialfrequency, the reduction of action potential height will opposeany stimulatory effect mediated by increased action potentialfiring and result in net inhibition of glucagon release. Thisis because exocytosis in both rodent and human α-cells issteeply voltage dependent. Measurements of cell capacitance(as an indicator of exocytosis) suggest that exocytosis decreasesby 75–85% [44] when the voltage of the electrical stimulusis reduced from +10 mV (corresponding to the peak of theaction potential) to −10 mV (figure 5C) [44].
Glucagon Secretion Depends on Activationof High-Voltage Activated Ca2+-ChannelsHuman α-cells contain several types of voltage-gated Ca2+-channel but P/Q-type Ca2+-channels appear particularlysignificant for glucagon secretion and their activity is maximalat zero mV. Other types of voltage-gated Ca2+-channel(including T- and L-type) open at more negative voltagesbut they are not as tightly linked to exocytosis as the P/Q-type channels in human α-cells. Lowered action potentialamplitude can accordingly be expected to principally reduceP/Q-type Ca2+-channel activity, whereas L-type Ca2+-channelactivity is less affected. Glucose and tolbutamide may thereforeparadoxically increase [Ca2+]i in α-cells (reflecting thestimulation of action potential firing and opening of L-andT-type Ca2+-channels) and yet inhibit glucagon secretion(because of reduced activation of the P/Q-type Ca2+-channels).Indeed, there is evidence for such a dichotomoy between[Ca2+]i and glucagon secretion in mouse α-cells [45].
Low Concentrations of Diazoxide ReverseInhibitory Effect of GlucoseAlthough it is technically difficult to demonstrate the signifi-cance of KATP-channel closure in the control of α-cell electricalactivity/glucagon secretion by (direct) measurements of chan-nel activity (because it is under strong tonic suppression), wecan test for their involvement using pharmacological agents.Diazoxide is a potent activator of KATP-channels [46–48]. Ifglucose acts by closing the few KATP-channels that remain active
Figure 6. KATP-channel activity and control of glucagon secretion.Glucagon secretion measured in the presence of increasing concentrationsof the KATP-channel activator diazoxide at 8.3 or 10 mM glucose inmouse (A) and human (B) pancreatic islets, respectively. The dashedhorizontal lines indicate insulin or glucagon secretion measured in theabsence of glucose. *p < 0.05 (or better) versus no diazoxide. Datamodified from [48]. (C) Schematic relationship between KATP-channelactivity and glucagon secretion. See main text for details.
in α-cell at low glucose concentrations (i.e. conditions associ-ated with stimulation of glucagon secretion), then it should bepossible to counteract the inhibitory effect of glucose by appli-cation of low concentrations of diazoxide. As shown in figure 6,1–2 μM diazoxide completely reverses the inhibitory effect of8–10 mM glucose on glucagon secretion in mouse (A) andhuman islets (B). The stimulation produced by 2 μM diazoxidedid not affect insulin secretion measured in parallel [48]. Whenhigher concentrations of diazoxide are used, the stimulatoryeffect of the KATP-channel activator on glucagon secretionis superseded by an inhibitory effect and at concentrations>100 μM, glucagon secretion is as low as in the presence of
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DIABETES, OBESITY AND METABOLISM review articleglucose alone (i.e. with no diazoxide added). This reflects thestrong activation of KATP-channels with resultant membranehyperpolarization and suppression of action potential firing.In control experiments conducted in the absence of glucosein mouse [48] and human islets (own unpublished), increas-ing concentrations of diazoxide dose-dependently inhibitedglucagon secretion with no sign of a stimulatory effect at lowconcentrations. This suggests that the effect of diazoxide isspecific and that it selectively stimulates glucagon secretionthat has been inhibited by glucose.
Glucagon Secretion From Islets IsolatedFrom Type-2 Diabetic DonorsAs discussed above, the defects of glucagon secretion associatedwith diabetes in man are twofold: too much glucagon being
secreted at high glucose and too little at low glucose [4,11,52].Indeed, glucagon secretion may be paradoxically increased dur-ing a glucose challenge [3]. To the best of our knowledge, noattempts have yet been made to correlate these in vivo observa-tions to the situation in vitro. We have examined the propertiesof six islet preparations from organ donors diagnosed with type-2 diabetes. It is of interest that five of these six preparationsresponded with stimulation rather than inhibition of glucagonsecretion when glucose was increased from 1 to 20 mM. Thedata are summarized in figure 7A. On average, glucose stimu-lated glucagon secretion by 47 ± 22% (n = 6; p < 0.05). In sixage- and BMI-matched ‘control’ islet preparations (i.e. fromdonors with no known diabetes), glucagon secretion at 20 mMglucose was inhibited by 34 ± 8% (n = 6; p < 0.01). The rateof glucagon secretion (expressed as a fraction of islet con-tent) averaged 1.1 ± 0.2%/h and 0.45 ± 0.12%/h (p < 0.05)in the non-diabetic and diabetic islets, respectively. Thus, theglucagon secretion defect we observe in α-cells/islets fromdonors with type-2 diabetes echoes that previously docu-mented in vivo [3]. In diabetic islets, 20 mM glucose stimulatedinsulin secretion 2.4 ± 0.5-fold (p < 0.01), less than 50% ofthe 5.1 ± 0.8-fold stimulation seen in islets from non-diabeticislets (figure 7B; p < 0.01). The lower stimulation index indiabetic islets (p < 0.01 compared to non-diabetic islets) is ingood agreement with that previously reported by others [53].
Islets from donor with type-2 diabetes contained onaverage 23 ± 4 ng/islet insulin (n = 6), 25% less than the32 ± 5 ng/islets (n = 6) seen in non-diabetic donors; an effectthat was not statistically significant. Glucagon content wasthreefold higher in islets from donors with type-2 diabetes;1949 ± 654 pg/islet vs. 588 ± 171 pg/islet (p ≈ 0.05). Thus, thelower fractional rate of basal glucagon secretion (at least in thisrather limited experimental series) is almost fully compensatedfor by the increased islet glucagon content.
The finding that insulin therapy corrects the hypersecretionof glucagon has been used to argue that the α-cell is underparacrine control of the β-cell. In this context, it is worthremembering that the paracrine control need not only (if at all)be exerted at the level of the minute-by-minute controlof glucagon secretion, insulin could also exert long-termeffects by affecting gene expression [54]. If insulin signalling isimpaired in diabetic islets (insufficient insulin being secreted,
Figure 7. Glucose stimulates glucagon secretion in islets from type-2 diabetic donors. (A) Glucagon secretion measured at 1 and 20 mM glucose ([Glc])in six islet preparations obtained from non-diabetic (ND) and type-2 diabetic (T2D) organ donors. Mean values ± s.e.m. Secretion has been normalized toislet glucagon (A) and insulin (B) contents. (C) Glucagon secretion in wildtype (black) and KATP-channel-deficient Kir6.2STOP mouse (grey) pancreaticislets measured at 0 or 20 mM glucose. *p < 0.05 (or better) versus 1 mM glucose.
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altered architecture of the islets or ‘α-cell insulin resistance’),this may affect the functional properties of the α-cells viachanges in the levels of key regulatory proteins. One interestingpossibility is that insulin signalling affects KATP-channel activityand/or expression in the α-cell. There is a precedent forthe former possibility in hypothalamic neurones [55] as wellas in β-cells [56]. In this context, it is of interest that theinversed glucagon response is also observed in KATP-channeldeficient (Kir6.2STOP) mice (figure 7C). It is possible that thestimulation of glucagon secretion seen in these mice reflect thesame mechanisms as that which produces the ‘stimulation’ ofglucagon secretion seen when glucose is increased from 5 to20 mM glucose (figure 1A). The finding that this stimulationis maintained in Kir6.2STOP islets suggests that this effectinvolves a KATP-channel-independent mechanism. Elucidationof the underlying mechanism may provide clues to theprevention of the glucose-induced stimulation of glucagonsecretion in type-2 diabetes.
Why Do Sulphonylureas Not InhibitGlucagon Secretion in Diabetic Patients—OrDo They?Given that glucagon secretion is under the control of KATP-channel and that tolbutamide inhibits glucagon secretion fromisolated islets from both rodent and human islets [48], itmay seem surprising that sulphonylureas therapy does notcorrect the hypersecretion of glucagon in type-1 diabetes [31];sulphonylureas have in fact been reported to stimulate glucagonsecretion [31,57,58]. By contrast, sulphonylureas have beenfound to inhibit glucagon secretion in both healthy [59]and type-2 diabetic subjects [60]. Banarer et al. [59] report
that tolbutamide inhibits the insulin/hypoglycaemia-inducedstimulation of glucagon secretion. This was interpreted to besecondary to intra-islet hyperinsulinaemia but the inhibitoryeffect of tolbutamide was only apparent when the plasmaglucose concentrations were ≤4.2 mM and when tolbutamidedid not produce a further stimulation of insulin secretion(cf. Figs. 5 and 6 in [59]) and the possibility that this effectreflects a direct effect on the α-cell should therefore beconsidered.
Effects of Insulin on Glucagon SecretionMice lacking insulin receptors in theα-cells have been generatedand the data emanating from studies on this model arevery instructive [61]. The increase in plasma glucagon levelsfollowing an intra-peritoneal arginine test is larger in micelacking insulin receptors in their α-cells, consistent with theidea that insulin (also released by arginine); possibly resultingfrom altered gene expression due to the lowered intra-isletinsulin levels exerts an inhibitory effect on glucagon secretion.In the knockout mice, infusion of exogenous insulin andthe associated hypoglycaemia resulted in a fivefold increasein plasma glucagon secretion. Interestingly, the stimulationof glucagon secretion in wildtype mice was only marginallyweaker (3.5-fold). In fact, the observation that insulin-inducedhypoglycaemia is a powerful stimulus of glucagon secretion inwildtype mice despite the presence of high circulating insulinlevels provides circumstantial evidence that the α-cells possessan intrinsic capacity to sense the fall in plasma glucose andthat this effect supersedes any inhibitory effect exerted byinsulin. As discussed earlier, we believe that this effect may bemediated by activation of KATP-channels. It would thereforebe very interesting to test whether the stimulation of glucagon
Figure 8. Model for the regulation of glucagon secretion. See main text for details.
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DIABETES, OBESITY AND METABOLISM review articlesecretion induced by hypoglycaemia in the insulin receptorknockout mice can be abolished by sulphonylureas.
Glucagon Secretion: Differential Rolesof Intrinsic and Paracrine RegulationLike insulin secretion, glucagon release is also influenced byamino and free fatty acids (FFAs). Thus, a cocktail of aminoacids (including arginine; [15]) and acute exposure to FFA arestrong stimuli of glucagon release [62]. Detailed knowledgeof the interactions between glucose and the amino acidsand/or FFAs in the control of glucagon secretion is essential tounderstand how the α-cell distinguishes between an elevationof plasma FFAs and amino acids following a mixed meal(when glucagon secretion should be suppressed) and when theybecome elevated during fasting/starvation by mobilization ofthe body’s depots (associated with stimulated glucagon release).
We propose a unifying hypothesis that integrates bothparacrine and intrinsic regulation of glucagon secretion.During hypoglycaemia (i.e. induced by exercise [63]) glucagonsecretion is enhanced because of slight activation of theKATP-channels with resultant firing of large-amplitude actionpotentials (figure 8A). An increase in glucose (by ingestion of aglucose-rich meal) inhibits glucagon secretion by closure of theKATP-channels. The resulting membrane depolarization leadsto a decreased amplitude of the α-cell action potential andreduced activation of voltage-gated P/Q-type Ca2+-channelswhich culminates in suppression of exocytosis (figure 8B).Following ingestion of a mixed meal (rich in glucose, aminoacids and lipids), glucagon secretion is switched off by acombination of KATP-channel closure (as in figure 8B) andactivation of paracrine inhibitory signalling (indicated by thearrow and minus sign) due to parallel stimulation of secretionin the neighbouring β- and δ-cells. The latter supersedes thestimulatory effects of amino and free fatty acids (figure 8C). Inthe fasted state (and during starvation), however, when plasmaconcentrations of free fatty and amino acids are increased bymobilization of bodily depots, the low plasma glucose levels, viaincreased KATP-channel activity and membrane repolarization,ensures that hormone release from β- and δ-cells is minimal.In the absence of inhibitory paracrine signals derived fromthese cells, the stimulatory effect of low glucose (mediatedby activation of KATP-channels) is amplified by the presenceof amino and free fatty acids (figure 8D), leading to strongstimulation of glucagon secretion.
The scenario outlined above suggests that the relativeimportance of intrinsic and paracrine regulation of glucagonsecretion depends on the physiological context. These con-siderations suggest that much of the published conflictingdata on the relative control of intrinsic and paracrine controlof glucagon secretion may reflect the different experimentalprotocols (single α-cells or intact islets, the glucose concen-trations employed and/or presence of other nutrients, in vitroor in vivo etc.). However, as we try to argue here, these dis-crepancies may be apparent and rather reflect different facetsof the metabolic regulation of glucagon secretion. Finally,the differences between rodent and human islets should notbe underestimated. We have already observed a number of
differences between mouse and human α-cells in terms ofboth the intrinsic [22,23], GABAergic paracrine [24–26] andincretin-mediated regulation of glucagon secretion [34,64].Data obtained in rodent islets should accordingly not beassumed to be applicable to the situation in man and it istherefore imperative that key observations are always validatedusing human islets.
AcknowledgementsThe studies on human islets were performed with ethicalconsent from the National Research Ethics Service Oxfordshire.Supported by the National Institute of Health Research (NIHR)Biomedical Research Centre (Oxford), the MRC and theWellcome Trust. PR is a Canada Excellence Research Chair(CERC).
Conflict of InterestsThe content is solely the responsibility of the authors and theyhave no conflicts of interest to disclose.
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