Invited Review

Glucose regulation of glucagon secretion

Erik Gylfe a,*, Patrick Gilon b

aDepartment of Medical Cell Biology, Uppsala University, BMC Box 571, SE-751 23, Uppsala, Swedenb Pole d’Endocrinologie, Diabete et Nutrition, Institut de Recherche Experimentale et Clinique,

Universite Catholique de Louvain, Brussels, Belgium

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2. Control of glucagon secretion during glucose counterregulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.1. Auto- and paracrine mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.2. Intrinsic a-cell mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.2.1. KATP channel-dependent glucose sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.2.2. Ca2+ store-dependent glucose sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.2.3. Other mechanisms for glucose sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3. Control of glucagon secretion during hyperglycaemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.1. Diminished inhibition of glucagon release in hyperglycaemia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.2. Paracrine induction of pulsatile glucagon release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

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a r t i c l e i n f o

Article history:

Received 28 November 2013

Available online 5 December 2013

Keyword:

Glucagon secretion

a b s t r a c t

Glucagon secreted by pancreatic a-cells is the major hyperglycemic hormone correcting acute

hypoglycaemia (glucose counterregulation). In diabetes the glucagon response to hypogly-

caemia becomes compromised and chronic hyperglucagonemia appears. There is increasing

awareness that glucagon excess may underlie important manifestations of diabetes. However

opinions differ widely how glucose controls glucagon secretion. The autonomous nervous

system plays an important role in the glucagon response to hypoglycaemia. But it is clear that

glucose controls glucagon secretion also by mechanisms involving direct effects on a-cells or

indirect effects via paracrine factors released from non-a-cells within the pancreatic islets.

The present review discusses these mechanisms and argues that different regulatory pro-

cesses are involved in a glucose concentration-dependent manner. Direct glucose effects on

the a-cell and autocrine mechanisms are probably most significant for the glucagon response

to hypoglycaemia. During hyperglycaemia, when secretion from b- and d-cells is stimulated,

paracrine inhibitory factors generate pulsatile glucagon release in opposite phase to pulsatile

release of insulin and somatostatin. High concentrations of glucose have also stimulatory

effects on glucagon secretion that tend to balance and even exceed the inhibitory influence.

The latter actions might underlie the paradoxical hyperglucagonemia that aggravates hyper-

glycaemia in persons with diabetes.

# 2013 Elsevier Ireland Ltd. All rights reserved.

* Corresponding author.

Contents available at ScienceDirect

Diabetes Researchand Clinical Practice

journal homepage: www.elsevier.com/locate/diabres

E-mail address: erik.gylfe@mcb.uu.se (E. Gylfe).

0168-8227/$ – see front matter # 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.diabres.2013.11.019

4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

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1. Introduction

Blood glucose-lowering insulin and -elevating glucagon are

the major hormones that act rapidly to maintain normogly-

caemia. Adrenaline contributes by inhibiting insulin and

stimulating glucagon secretion and by directly mimicking

the glucose-elevating action of glucagon. In diabetes, lack or

inappropriate secretion of insulin is the generally accepted

cause of hyperglycaemia, but Unger and Cherrington [1]

recently argued that ‘‘glucagon excess, rather than insulin

deficiency, is the sine qua non of diabetes’’. This provocative

statement illustrates an increasing awareness that glucagon

underlies important manifestations of diabetes. However,

glucagon dysregulation is not restricted to excessive release of

the glucose-elevating hormone that aggravates hyperglycae-

mia in persons with diabetes. An acutely more dangerous

aspect is that glucose counterregulation is compromised,

since glucagon secretion is not appropriately stimulated by

hypoglycaemia [2].

The hypoglycaemic glucose concentration range may seem

most appropriate for controlling glucagon secretion. However,

the hyperglucagonemia in diabetes may reflect a stimulatory

component in the effect of high glucose concentrations on

glucagon release [3,4] that indicates a physiological role of the

hormone also under hyperglycaemic conditions. In the

present review we therefore discuss regulation of glucagon

secretion by glucose concentrations ranging from those

characterizing hypo- to hyperglycaemia.

Whereas research during the last decades have resulted in

considerable understanding of molecular components by

which pancreatic b-cells recognize glucose and how subse-

quent signalling leads to insulin release [5], there are

fundamentally different ideas how glucose regulates gluca-

gon secretion. It has often been claimed that the nervous

system plays the major role in the control of glucagon

secretion during hypoglycaemia. Control via the autonomic

nervous system depends on hepatoportal and brain sensors.

There are three major autonomic inputs to the a-cells: the

sympathetic system secreting mainly noradrenaline, the

parasympathetic system secreting mainly acetylcholine and

the adrenal medulla releasing adrenaline [6,7]. The role of

pancreatic islet innervation in the control of glucagon

secretion should however be relativized since recent obser-

vations have shown that human islets are much less

innervated than rodent islets [8]. It is also clear that glucagon

secretion is regulated by glucose also without neural influ-

ence in the perfused pancreas and in isolated islets of

Langerhans. The present focus will be on intra-islet regula-

tion of glucagon release. This regulation has been proposed to

involve auto and paracrine interactions between a-cells and

the other islet cell types as well as a-cell intrinsic mecha-

nisms. As will become evident in the present review it is likely

that such processes partake in the regulation of glucagon

release, and that their relative importance depends on the

prevailing glucose concentration.

2. Control of glucagon secretion duringglucose counterregulation

2.1. Auto- and paracrine mechanisms

Secretion is an energy-requiring process, and from that point

of view it is logical that insulin release is stimulated when the

b-cells are energized by glucose. Glucagon secretion should be

optimal under less favourable energetic conditions, since

release is stimulated by lack of glucose. Positive feedback of

secreted factors may help to provide an adequate response

under such conditions. Glucagon has for example been found

to amplify its own secretion by raising cAMP [9,10] in the a-

cells. Moreover, glutamate co-released with glucagon was

reported to act on a-cell AMPA/kainate receptors to potentiate

glucagon secretion by raising the cytoplasmic Ca2+ concentra-

tion ([Ca2+]i) [11]. Human a-cells release acetylcholine in

parallel with glucagon [8] and this transmitter may be involved

in a similar positive feedback loop.

So far all paracrine mechanisms proposed to control

glucagon secretion from the a-cells involve release of

inhibitory factors from the b- and d-cells. In this context

considerable emphasis has been made on islet cytoarchitec-

ture and the direction of blood flow within the islet. In rodent

islet with b-cells predominantly located in the islet core and a-

and d-cell in the periphery [12] blood flow from the centre to

the periphery [13] may facilitate the paracrine control of the a-

cells. However, paracrine control does not necessarily depend

on capillary transport since diffusion in the interstitial space

may suffice. Indeed, the importance of the islet microcircula-

tion has been questioned in human islets where the different

cell types occur without particular order [14]. Since glucose

stimulates insulin release and insulin inhibits glucagon

secretion it has been proposed that inhibition of the a-cells

is secondary to stimulation of the b-cells. Insulin was the first

putative mediator of this inhibition [15] but later studies have

favoured Zn2+ that is co-released with insulin from the

secretory granules [16], g-aminobutyric acid (GABA) [17] and

its metabolite g-hydroxybutyric acid (GHB) [18]. Studies with

ZnT8 knockout mice in which Zn2+ is no longer stored in b-cell

secretory granules have however questioned the role of Zn2+

[19,20]. It should be emphasized that two routes of release of

paracrine factors from b-cells are possible: a Ca2+-dependent

release from secretory vesicles or a Ca2+-independent release

via plasma membrane transporters. The first process of

release makes unlikely that insulin is involved in the glucagon

response to hypoglycaemia because insulin secretion from

islets essentially remains basal when glucagon release

becomes maximally inhibited as the glucose concentration

is raised in the 0–7 mM range (Fig. 1). However, it might play a

role at higher glucose concentrations (see below). The second,

Ca2+-independent process of release is compatible with a

paracrine influence of b-cell-derived factors on glucagon

secretion in response to hypoglycaemia. GABA can be released

from b-cells by both routes. Indeed, it is accumulated mainly

Fig. 1 – Dose response relationship for glucose-regulated

secretion of glucagon compared to that of insulin (left

panel) and somatostatin (right panel). Batches of mouse

islets were incubated for 60 min with glucose

concentrations ranging from 0 to 30 mM. Glucagon

secretion is expressed in per cent of that stimulated by

absence of glucose whereas insulin and somatostatin

secretion are expressed in per cent of that stimulated by

30 mM glucose. Mean values W SEM for 8 experiments.

Observations from Vieira et al. [26] have been expanded

with previously unpublished secretion data at 25 and

30 mM glucose from the same experiments. Note that

glucagon secretion at >20 mM glucose exceeds that

stimulated by the absence of the sugar.

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in small synaptic-like microvesicles, which undergo Ca2+-

dependent exocytosis like insulin granules [21]. GABA is also

extruded by membrane transport. It has been suggested that

interstitial GABA tonically activates GABAA receptors in a-cells

[22]. However, its involvement in the glucagonostatic effect of

glucose may be questioned because 20 mM glucose has been

found to reduce both the content and the total GABA release

from b-cells [23]. Studies of human islets have indicated that

the glucose-induced reduction of GABA is associated with

increased content and release of GHB, which inhibits glucagon

secretion by putative GHB receptors in a-cells [18].

Somatostatin is a potent inhibitor of both insulin and

glucagon secretion and was early proposed to be a paracrine

regulator of glucagon secretion [24] mediating inhibition

during hyperglycaemia [25]. Fig. 1 shows that somatostatin

secretion increases with glucose concentration in parallel

with inhibition of glucagon release when glucose is raised in

the 0–7 mM range consistent with a role of somatostatin also

in glucose counterregulation. However, studies in 3 different

laboratories have shown that blockade of the a-cell somato-

statin receptors [26] or interference with somatostatin signal

transduction by pertussis toxin exposure [19,27] increase

glucagon secretion but does not prevent inhibition by

glucose. The d-cells apparently exert static control of the

a-cells but somatostatin does not seem to be the major

mediator of inhibition of glucagon release after recovery

from hypoglycaemia.

An argument for paracrine regulation of glucagon release is

that some studies (but not all) reported that glucose increases

the electrical activity [28] and raises [Ca2+]i [29] in isolated rat

a-cells and stimulates glucagon release from purified rat and

mouse a-cells by mechanisms that mirror those in b-cells

[29,30]. Intra-islet interactions may therefore be required for

glucose inhibition of glucagon release. However, in view of the

abovementioned arguments those interactions may be per-

missive rather than directly regulatory and/or operate in the

higher range of glucose concentrations.

2.2. Intrinsic a-cell mechanisms

Since glucose regulates glucagon, insulin and somatostatin

release and the hormone-secreting a, b and d-cell cells develop

from a common progenitor [31] it seems plausible that the

different types of islet cells may share features of glucose

recognition. The b-cell is by far the best-characterized cell

type. Glucose is rapidly taken up by facilitated diffusion by the

high Km GLUT2 transporter in rodent and the low Km GLUT1 in

human b-cells [32] but the significance of this difference in

unclear. Glucose phosphorylation is dominated by the rate-

limiting high Km enzyme glucokinase (more than 90% in intact

islets) [33,34]. During subsequent glycolysis most of the

pyruvate is further metabolized by the mitochondria [35].

The resulting increase of the cytoplasmic ATP/ADP ratio closes

ATP-dependent K+ (KATP) channels leading to depolarization

that opens voltage-dependent L-type Ca2+ channels and the

resulting rise of [Ca2+]i is the main trigger of insulin release [5].

Rodent a-cells essentially express the low Km GLUT1 and

glucose transport is about 10-fold slower than in the GLUT2

expressing b-cells, but still 5-fold higher than a-cell glucose

utilization [36]. The glucokinase activity in a-cells is similar to

that in b-cells and has been attributed a role as metabolic

glucose sensor [34]. There is also some expression of the low

Km hexokinase, which may be involved in the sensing of very

low sugar concentrations since it becomes saturated by 1 mM

glucose [34]. Whereas the glycolytic flux is comparable in b-

and a-cells [36], it is essentially aerobic in b- and anaerobic in

a-cells [35]. In accordance with such a difference glucose

induces much smaller changes in cytoplasmic ATP [16,37],

FAD [38] and NAD(P)H [39] in a- than in b-cells. Metabolism is

nevertheless essential for glucose recognition since glucose-

inhibited glucagon release is mimicked by a glucokinase

activator and secretion is not affected by a non-metabolizable

glucose transport analogue [19].

In the absence of glucose a-cells are electrically active and

show Ca2+ oscillations. Rise of [Ca2+]i is generally believed to

trigger exocytosis. The action potentials start at more negative

potentials than in the b-cells due to presence of T-type Ca2+

channels that activate at potentials as low as �60 mV and

tetrodotoxin (TTX)-sensitive Na+ channels, which activate at

potentials more positive than �30 mV [40,41]. Like b-cells the

action potentials in a-cells open high voltage-activated L-type

Ca2+ channels as well as N-type Ca2+ channels [42]. However,

the identification of the latter type of channel was based on an

unspecific blocker and later studies have instead indicated

involvement of P/Q channels [43]. Together the L- and N- or P/

Q-type channels contribute to most of the Ca2+ entry into the

a-cells. The relationship between [Ca2+]i and secretion is less

Fig. 2 – Model of glucagon secretion implicating that only a

small fraction of the entering Ca2+ triggers hormone

release and that sustained depolarization inhibits

secretion by reducing action potential amplitude. The

upper left part shows action potentials and the ion

permeabilities that contribute to the different phases and

the lower panel illustrates these channels in an a-cell.

Under stimulatory low glucose conditions the T-type Ca2+

channels fire spontaneously from about -60 mV and cause

a depolarization (c#) that activates TTX-sensitive Na+

channels (Nav), which in turn activate L-type and briefly P/

Q-type Ca2+ channels (red peaks of the action potentials).

The P/Q type channels are more important for secretion

than the L-type channels since the P/Q channels are

located closer to the secretory granules. Repolarization

depends on activation of voltage-dependent K+ channels

(Kv). Glucose inhibits glucagon secretion by slight

sustained depolarization after closing the last few open

KATP channels (not shown). The depolarization

preferentially inactivates the Nav channels thereby

preventing Ca2+ influx through the P/Q channels (upper

right) and glucagon release.

Illustration adapted from Rorsman et al. [43].

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clear in a- than in b-cells. Although L-type channels dominate

in rodent a-cells (80%) and mediate most Ca2+ entry during

action potentials, specific inhibitors of these channels have

little effect on glucagon secretion whereas blocking the non-L-

type channels (20%) has little effect on [Ca2+]i but inhibits

secretion as efficiently as glucose elevation to 6 mM [42–44].

The proposed explanation for this discrepancy is that the non-

L-type channels (presumably P/Q) are closely associated with

the secretory granules whereas the L-type channels are not

(Fig. 2). However, in the presence of adrenaline, which

depolarizes the a-cells and mobilizes Ca2+ from the ER

[45,46] entry of extracellular Ca2+ through the L-type channels

will be the most important trigger of secretion [43,47]. The

situation is not less complicated in human a-cells. Although

the granule-associated P/Q-channels dominate (70%) over the

L-channels (20%) and are responsible for most of the

exocytosis, the P/Q channels open very briefly and only

mediate a small fraction of the Ca2+ entry [48]. An attractive

aspect of such Ca2+ regulation is that it can explain why

glucose inhibition of glucagon secretion is associated with

rather modest reduction of [Ca2+]i signalling [39,46,49,50].

2.2.1. KATP channel-dependent glucose sensingThe most cited hypothesis for intrinsic glucose regulation of

glucagon secretion attributes a b-cell-like central role of the

KATP channels in the regulation of glucagon release [43]. It is

postulated that rise of glucose increases ATP and depolarizes

the a-cells by closing KATP channels. However, unlike in the b-

cells this depolarization results in inhibition of secretion. The

reason for this paradox is that a-cells display features that are

not found in b-cells. Indeed, the KATP channel conductance is

extremely low already when the a-cells are exposed to very

low concentrations of glucose. Moreover, activation of high-

threshold voltage-dependent channels triggering the Ca2+-

dependent exocytosis (mainly P/Q-type, but also L-type)

occurs only if low-threshold voltage-dependent channels (T-

type Ca2+ and Na+ channels) are opened, and these low-

threshold voltage-dependent channels undergo inactivation

upon sustained depolarization [39,40]. Hence, complete

closure of KATP channels in response to glucose-induced

ATP formation causes a small depolarization that is insuffi-

cient to open voltage-dependent L and P/Q type of Ca2+

channels but this sustained depolarization inactivates the low

threshold T-type Ca2+ and Na+ channels. Therefore the action

potential amplitude becomes sufficiently reduced to fail to

activate secretion-triggering Ca2+ influx through P/Q channels

although the L-channels still contribute to [Ca2+]i elevation

(Fig. 2). In this model glucagon secretion consequently

depends on the amplitude and frequency of the action

potentials. The model implies that the action potentials

generating glucagon secretion occur only in a narrow window

of membrane potentials. They are prevented at potentials

more negative than those activating T-type Ca2+ channels and

also by sustained depolarization to potentials that inactivate

the T-type Ca2+ and in particular the Na+ channels. In

accordance with the model, KATP channel closure with

sulfonylurea or slight depolarization by elevation of the K+

concentration mimic the inhibitory effect of glucose [44,51]

and slight hyperpolarization with low concentrations of KATP

channel-activating diazoxide have been found to reverse

glucose inhibition of glucagon secretion whereas more

pronounced hyperpolarization inhibits secretion [44,52].

Moreover, the inhibitory effect of glucose is lost or attenuated

after functional knockout of the KATP channels [51,53,54].

Despite some enticing experimental support for the KATP

channel-centred hypothesis for glucose regulation of glucagon

secretion, there are also conflicting evidence, and the concept

Fig. 3 – The store-operated model for glucose regulation of

glucagon release. Under hypoglycaemic conditions (left)

the SERCA pump is poorly energized and the ER contains

little Ca2+, a situation that activates store-operated

channels (SOC). The resulting cation influx depolarizes

(c#) the a-cell and opens voltage-dependent Ca2+ channels

(VDCC). The subsequent influx of Ca2+ triggers glucagon

release. In normoglycaemia the SERCA pump is properly

energized and the ER is filled with Ca2+, thus shutting off

the depolarizing store-operated cation influx and Ca2+

entry through VDCC as well as glucagon release.

Illustration based on Liu et al. [46] and Vieira et al. [26].

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is not unanimously accepted. Whereas glucose elevation tends

to decrease [Ca2+]i in a-cells, there are several studies reporting

that complete KATP channel closure by sulfonylureas causes b-

cell-like [Ca2+]i elevation rather than an expected reduction

[26,39,46,55], and at glucose concentrations that inhibit gluca-

gon release, sulfonylurea becomes stimulatory [19]. Moreover,

glucose retains its inhibitory effect on glucagon secretion when

the KATP channels are completely closed in the presence of high

concentrations of sulfonylurea [26], a situation associated with

lowering of [Ca2+]i [26,39]. Low diazoxide concentrations that

slightly hyperpolarize and reverse glucose inhibition of gluca-

gon release from batch-incubated islets [44,52] fail to reverse

glucose inhibition of glucagon secretion from perifused islets

[19]. At high concentrations of diazoxide, which maximally

open the KATP channels to hyperpolarize the a-cells and inhibit

glucagon release, glucose can still inhibit secretion further

[3,19]. Glucose retains an inhibitory effect on glucagon secretion

in some studies of KATP channel knockout islets [6,19] and this

effect is promoted by disruption of somatostatin signalling,

which eliminates its static inhibitory effect on glucagon

secretion [19].

An important aspect of the KATP channel hypothesis is that

glucose depolarizes the a-cell, but even this is controversial.

Measurements with invasive electrophysiological techniques

have indicated both depolarizing [51] and hyperpolarizing

[40,56–58] effects. It may seem strange that a fundamental

aspect like the glucose effect on membrane potential is not yet

settled, but it may be related to the high input resistance of the

relatively small a-cells [40]. Therefore the potential is very

sensitive to small currents, some of which may be related to

the invasive approach. At variance with the KATP channel

hypothesis membrane potential measurements with dye-

based non-invasive technique indicate that glucose hyperpo-

larizes the a-cell [46,59].

2.2.2. Ca2+ store-dependent glucose sensingSince it is generally assumed that glucagon secretion is

triggered by voltage-dependent Ca2+ entry it seems most

straightforward if lack of glucose were to depolarize the a-cell,

as is often observed [40,46,56–59]. Glucose sensing models

based on a hyperpolarizing action of the sugar do not exclude

involvement of a similar ion channel repertoire for action

potential generation as described above. The first detectable

effect of glucose elevation on [Ca2+]i in b-cells is a lowering [60]

that coincides with early depolarization [61] and inhibition of

insulin release [62]. When the depolarization is sufficient to

open voltage-dependent Ca2+ channels there is a [Ca2+]ielevation [61] that triggers first phase secretion [63]. The

initial [Ca2+]i lowering is due to ATP-energized Ca2+ seques-

tration into the endoplasmatic reticulum (ER), since glucose-

sequestered Ca2+ can be mobilized by inositol 1,4,5-trispho-

sphate (IP3)-generating agonists [64] and is prevented by

blocking the sarco(endo)plasmatic Ca2+ ATPase (SERCA-pump)

[61]. The same mechanism was identified in guinea-pig a-cell

where adrenaline was found to mobilize glucose-incorporated

Ca2+ and their opposite effects on [Ca2+]i were proposed to

explain glucose inhibition and adrenaline stimulation of

glucagon release [65]. A problem with this concept is that

the ER has a limited Ca2+-sequestering capacity implying that

Ca2+ uptake and release can only account for temporary

inhibition and stimulation. This is where the store-operated

mechanism comes in, since Ca2+ store depletion triggers a

depolarizing current that at least is partly carried by Ca2+

influx. The store-operated current is small and alone it only

causes a modest [Ca2+]i elevation in both b- or a-cells

[46,66,67]. Activation of this current by SERCA inhibition

marginally elevates basal [Ca2+]i in b-cells [61] whereas a-cells

respond with a pronounced [Ca2+]i elevation involving also

voltage-dependent Ca2+ entry [46] resulting in glucagon

release [26]. The reason for the higher sensitivity of the a-

cells is apparently that their high input resistance [40] makes

the membrane potential more sensitive to small currents that

thereby can trigger voltage-dependent Ca2+ influx.

In b-cells the store-operated Ca2+ influx is inversely related

to the Ca2+ content of the ER in a graded manner [67]. a-Cell

glucose sensing via the store-operated mechanism implies that

under hypoglycaemic conditions the ER contains little Ca2+ and

that the store-operated pathway depolarizes the a-cells to

stimulate glucagon release (Fig. 3). At normoglycaemia the

SERCA pump becomes sufficiently energized to fill the ER and

shut off this stimulatory cascade. It is therefore pertinent to

note that there is a major difference between a- and b-cells with

regard to the glucose sensitivity of ER Ca2+ filling. Whereas 8–

20 mM glucose is required to fill the ER of b-cells [64,68,69]

maximum is reached at 3 mM in the a-cells [46]. The different

sensitivities were reinforced when analysing the kinetics of the

ER Ca2+ sensor STIM1 and the plasma membrane Ca2+ channel

protein Orai1, which are the molecular components of the

store-operated pathway. In both a- and b-cells emptying of ER

Ca2+ induces STIM1 translocation from the ER to the PM where it

co-clusters with Orai1 to activate the store-operated current.

Glucose reverses this process by stimulating re-translocation of

STIM1 to the ER in a graded fashion, thus shutting of the store-

operated current [70]. Whereas this process is saturated by 11–

20 mM glucose in b-cells, only 3 mM is required in a-cells. The

store-operated pathway in a-cells is consequently controlled in

a concentration range that is relevant for glucose counter-

regulation. An attractive feature of the Ca2+ store-dependent

Fig. 4 – Increase of the glucose concentration from 3 to

20 mM induces pulsatile release of insulin, glucagon and

somatostatin from a perifused batch of 15 human

pancreatic islets. The insulin and glucagon pulses are

synchronized whereas the glucagon pulses are in opposite

phase. The shaded areas show secreted hormone mass

indicating time-average stimulation of insulin and

somatostatin secretion and inhibition of glucagon release.

Despite the time-average inhibition of glucagon release,

the peaks of the pulses exceed secretion at 3 mM glucose.

Illustration adapted from Hellman et al. [79].

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 0 3 ( 2 0 1 4 ) 1 – 1 06

glucose sensing is that glucose inhibition and adrenaline

stimulation of glucagon release can be explained by their

opposite effects on the calcium content of the ER that controls

the depolarizing store-operated current.

2.2.3. Other mechanisms for glucose sensingTwo less explored models are also based on a hyperpolarizing

action of glucose, which is assumed to reflect energization of

the hyperpolarizing, electrogenic Na+/K+ pump [71], or a

glucose-induced a-cell swelling [72] with subsequent activa-

tion of Cl� influx through volume-regulated channels [73] and/

or the cystic fibrosis transmembrane conductance regulator

protein [74]. Whereas rise of ATP is essential for all the

abovementioned models there is one that instead focuses on

the concomitant drop in AMP, which is assumed to inhibit

glucagon release by inactivating AMP-dependent protein

kinase (AMPK) [75]. Glucose thus inhibited AMPK activity in

a clonal a-cell line and pharmacological or molecular genetic

activation of AMPK stimulated glucagon secretion whereas

AMPK inactivation prevented [Ca2+]i elevation and glucagon

release at low glucose concentrations. AMPK may partly act

independently and downstream of Ca2+, which would be

consistent with a proposal that glucose activates a Ca2+-

independent inhibitory pathway [76].

3. Control of glucagon secretion duringhyperglycaemia

3.1. Diminished inhibition of glucagon release inhyperglycaemia

Consistent with its important role in glucose counterregula-

tion, glucagon secretion is maximally inhibited by 7–8 mM

glucose [3,4,26,44,77]. There are several reports that the

inhibitory action declines significantly at higher concentra-

tions of the sugar in both mouse [3,4,26,44] and human islets

[4]. Concentrations above 20 mM even stimulate secretion

from mouse islets as shown in Fig. 1 and reported previously

[3] but not in other studies of mouse [19] and rat [77] islets.

Additional evidence for a stimulatory effect of high glucose

concentrations come from studies of pulsatile glucagon

release with peaks of secretion that exceed secretion

stimulated by low concentrations of the sugar (Fig. 4)

[78,79]. Diminished inhibition may either reflect a peculiar

glucose dependence of the inhibitory mechanism or that

inhibition is counteracted by a stimulatory effect of high

glucose concentrations. The presence of a strong stimulatory

component might overcome the maximal inhibition by 7–

8 mM glucose as well as the expected additional inhibition

from the abovementioned paracrine factors when b- and d-cell

secretion become stimulated by high glucose concentrations.

The nature of this stimulatory component is challenging. One

possibility would be that the KATP channels are coupled to

stimulation of glucagon secretion by mechanisms that mirror

those in b-cells as observed in purified a-cells [29,30]. However,

whereas full KATP channel activation with diazoxide hyper-

polarizes the a-cells and reduces glucagon secretion in the

absence of glucose, the stimulation observed when increasing

the glucose concentration in the 7–30 mM range is actually

somewhat enhanced [3]. The latter effect of diazoxide may

result from reduced secretion of inhibitory factors from b- and

d-cells, and the glucose-stimulated glucagon secretion from

diazoxide-hyperpolarized a-cells indicates involvement of a

Ca2+ independent process. Maybe this effect involves cAMP,

which is another stimulatory messenger of glucagon secretion

[9,42,45], since 20–30 mM glucose has been found to cause

pronounced elevation of cAMP in a-cells [10]. The reduced

glucose inhibition of glucagon secretion observed at high

glucose concentrations deserves considerable attention since

it likely explains the complicating hyperglucagonemia in

persons with diabetes hyperglycaemia.

3.2. Paracrine induction of pulsatile glucagon release

Under hyperglycaemic conditions when both b- and d-cell

secretion is stimulated (Fig. 1) it is likely that inhibitory

paracrine factors influence glucagon secretion, as exemplified

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 0 3 ( 2 0 1 4 ) 1 – 1 0 7

by the observation that selective b-cell activation causes

inhibition of glucagon secretion [80]. Other convincing

evidence for paracrine regulation during hyperglycaemia

comes from studies of islet hormone secretion kinetics from

mouse and human islets [78,79]. As illustrated in Fig. 4,

secretion of insulin, somatostatin and glucagon proceed at

stable rates in the presence of 3 mM glucose but after increase

to 20 mM the secretion of all the hormones become strongly

pulsatile with insulin and somatostatin in phase and glucagon

in antiphase. It is well known how pulsatile insulin release is

generated. At low glucose concentrations [Ca2+]i is low and

stable in individual b-cells and exposure to high concentra-

tions of the sugar induce slow concomitant oscillations of

[Ca2+]i and [cAMP]i that trigger and amplify pulsatile insulin

release, respectively [81]. Due to gap-junction coupling the

[Ca2+]i oscillations become synchronized among b-cells [82] to

generate pulsatile insulin release from entire pancreatic islets

[83,84]. Paracrine factors like ATP released together with

insulin also act synchronizing even between isolated islets [85]

explaining how pulsatile insulin secretion with 5–8 min

periodicity can be observed from batches of perifused

pancreatic islets [78,79].

Although individual a- and d-cells often show [Ca2+]ioscillations in 3 mM glucose [50,86] hormone secretion from

whole islets occurs without pulsatility [78,79]. The obvious

reason is that neither a- nor d-cells are electrically coupled,

and they show asynchronous behaviour within islets [87]. The

concomitant occurrence of pulsatile insulin, glucagon and

somatostatin secretion at high glucose concentrations indi-

cates that paracrine factors originating from the b-cells

determine the pulsatility of somatostatin and glucagon

secretion. Since the insulin and somatostatin pulses are in

phase (Fig. 4) the b-cells must secrete a somatostatin-

stimulating factor. ATP, which is involved in b-cell synchro-

nization between perifused islets and probably in neural islet

coordination in vivo [85], may explain the synchronization also

of d-cells, since P2 purinergic agonists are potent stimulators of

somatostatin secretion [88]. Pulsatile glucagon secretion

occurs in opposite phase with nadirs corresponding to

coinciding peaks of insulin and somatostatin (Fig. 4). It seems

likely that somatostatin as well as abovementioned inhibitory

b-cell factors contribute to this phase relationship. A similar

relationship between pulsatile release of insulin and glucagon

is observed in healthy human subjects [89] in which pulsatile

delivery of insulin and glucagon has greater effects in

modulating endogenous glucose production than continuous

infusion [90]. Although glucose elevation from 3 to 20 mM

inhibits time-average glucagon release from isolated islets, the

peaks of secretion actually exceed stimulated secretion at the

lower sugar concentration emphasizing a stimulatory compo-

nent in the glucose effect. A consequence of insulin and

glucagon pulses in opposite phase is that the insulin/glucagon

ratio, which determines glucose storage and release from the

liver, will vary at least 20-fold [78,79]. This should mean that

during hyperglycaemia, the liver rapidly switches between

storing and releasing glucose although the net effect is

storage. We can only speculate why energy is spent on such

a cyclic process. It may perhaps promote even distribution of

glycogen in the liver rather than preferential storage in cells

that are first reached by the hormones.

4. Concluding remarks

Much confusion about glucose regulation of glucagon

secretion may result from research designs that fail to clarify

how different processes become involved in a glucose

concentration-dependent manner. In this review we have

argued that a-cell intrinsic glucose sensing determines

glucagon release during recovery from hypoglycaemia, and

that inhibitory paracrine factors become important in

hyperglycaemia. Nevertheless, glucagon secretion is less

inhibited or even enhanced in hyperglycaemia due to the

presence also of stimulatory effects of the sugar. In

hyperglycaemia glucagon and insulin release become pulsa-

tile with the peaks of glucagon coinciding with the nadirs of

insulin. This characteristic phase relationship is lost in

human prediabetes [89], which should reduce glucose

elimination by the liver.

Another problem in diabetes is the impaired glucose

counter regulatory response with inappropriate glucagon

secretion during hypoglycaemia [2]. Understanding and

correction of the underlying mechanism may not only prevent

life-threatening hypoglycaemia in insulin- or sulfonylurea-

treated patients but open for a novel therapeutic principle

with glucagon fine-tuning normoglycaemia in persons with

diabetes intentionally treated with slight overdoses of insulin

or insulin-elevating drugs.

Conflict of interests

We have no conflict of interests.

Acknowledgements

The authors are supported by the Swedish Research Council

(55X-06240), the Swedish Diabetes Association, the Fonds de la

Recherche Scientifique Medicale (Brussels, grant 3.4554.10)

and the Actions de Recherche Concertees (ARC 13/18-051)

from the General Direction of Scientific Research of the French

Community of Belgium. P. Gilon is Research Director of the

Fonds National de la Recherche Scientifique, Brussels.

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