Role of mitochondria in metabolism-secretion coupling of insulin release in the pancreatic beta-cell

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Role of mitochondria in metabolism-secretion coupling of insulin release in the pancreatic beta-cell



The control of insulin secretion from the pancreatic beta-cell involves a complex cascade of events in which the mitochondria play a central role. In the consensus model this role is essentially restricted to the production of ATP promoting membrane depolarisation and a rise in cytosolic [Ca2+]. Evidence for the generation of an additional mitochondrial factor implicated in metabolism-secretion coupling is provided in this review. Although not yet identified, the formation of this putative factor requires an increase of [Ca2+] in the mitochondrial matrix together with a supply of carbons to the tricarboxylic acid (TCA) cycle. In this model, calcium activates matrix dehydrogenases, in particular those of the TCA cycle.

This enables the synthesis of the mitochondrial factor from the TCA cycle intermediates.

Experimental evidence gathered in permeabilised cells largely supports this model.

MAECHLER, Pierre, WOLLHEIM, Claes. Role of mitochondria in metabolism-secretion coupling of insulin release in the pancreatic beta-cell. BioFactors, 1998, vol. 8, no. 3-4, p. 255-62

PMID : 9914827

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BioFactors 8 (1998) 25 5—262


Role of mitochondria in

metabolism-secretion coupling of

insulin release in the pancreatic ,13-cell

Pierre Maechler and Claes B. Wollheim*

Division of Clinical Biochemistry, Department of Internai Medicine, University Medical Centre, CH-1211 Geneva 4, Switzerland

Received 8 July 1998

Abstract. The control of insulin secretion from the pancreaticfi-ecu involves a complex cascade of events in which the mi tochondria play a central role. In the consensus model this role 15 essentially restricted to the production of Ai? promoting membrane depolarisation and a rise in cytosolic [Ca2j. Evidence for the generation of an additional mitochondrial factor im plicated in metabolism-secretion coupling is provided in this review. Although not yet identified, the formation of this putative factor requires an increase of [Ca2j in the mitochondrial matrix together with a supply of carbons to the tricarboxylic acid (TCA) cycle. In this model, calcium activates matrix dehydrogenases, in particular those of the TCA cycle. This enables the synthesis of the mitochondrial factor from the TCA cycle intermediates. Experimental evidence gathered in permeabilised cells largely supports this model.

1. Introduction

Blood glucose homeostasis is mainly controlled by insulin through its secretion by the pancreatic /3-cell and its action on target tissues ([47], and references therein). The most important physiological nutrient stimuli of insulin release are glucose and leucine [35]. The action of these secretagognes is po tentiated by gastrointestinal hormones, which activate either adenylate cyclase or phospholipase C [47].

In contrast to die receptor-mediated signal transduction triggered by the hormones, glucose and leucine generate signais following their metabolism by die f3-cell. Glucose enters die ~3-cell by facilitated diffu sion and undergoes glycolysis before its final oxidation in die mitochondria, whereas leucine is directly metabolised by die mitochondria.

Non-insulin-dependent-diabetes-meilitus (NIDDM), or type II diabetes, is a polygenic disease char acterised by impaired insulin secretion [32] often combined with insulin resistance [17]. A rare subform of NIDDM referred to as maternally inherited diabetes and deafness (MIDD) has been linked to muta tions in die mitochondrial genome. These mutations involve deletions or point mutations such as that in ~ at position 3243 also giving rise to mitochondriai encephaiomyopathy, lactic acidosis and stroke-lilce syndromes (MELAS) [22]. In MIDD diere is a progressive impairment of insulin secre tion, which may eventually lead to severe insulin deficiency reminiscent of type I (insulin-dependent) diabetes.

‘Correspondence to: Dr Claes B. Wollheim, Division de Biochimie Clinique, Centre Médical Universitaire, 1211 Geneva 4, Switzerland. Tel.:++41 2270255 48; Fax:++41 22702 55 43; E-mail:

095 1-6433/9848.00 © 1998—IOS Press. AIl rights reserved


256 R Matchier and CB. Wo!!heim /Mitochondria and insulin secretion

2. SignaI generation in insulin secretion

In the pancreatic /3-celi, mitochondrial metaboiism plays a pivotai role in the generation of signais coupling glucose recognition to insulin secretion [11,27,30,35]. Metabolism of nutrient stimuli in the


ceil causes the closure of ATP-sensitive K+ (KATP) channels and depolarisation of the plasma membrane potential [38]. This ieads to Ca2+ influx through voltage-gated Ca2+ channels and a rise in cytosolic


([Ca2j~) [45,47]. The increase in [Ca2+]c is the main trigger of exocytosis, the process by which the insulin containing secretory granules fuse with the plasma membrane (4,38,47]. Ca2+, the universal second messenger, controis numerous cellular functions inciuding mitochondriai metabolism [15,28]. An increase in [Ca2+]c is relayed to the mitochondria with the consequent rise in matrix Ca2+ concentration ([Ca2t]m) [36,37]. This elevation in [Ca2+]m ieads to stimulation of Ca2+~sensitive NADH-generating dehydrogenases [6,14,28,34,39,43]. The latter process potentiates the generation of NADH which trans fers reducing equivalents to the respiratory chain, thereby ensuring adequate ATP synthesis to balance the augmented energy needs of celI activation [28]. Incubation of islet fi-cells in the absence of extra cellular Ca2+ results in a decrease of glucose oxidation without an apparent change in the ATP/ADP ratio [43]. This emphasises the observation that a tise in [Ca2+]m is not necessary for the initial signal generation by the mitochondria in response to glucose, i.e., the increases in NAD(P)H [33] and in the ATP/ADP ratio [31]. The ensuing elevation in [Ca2+]c augments [Ca2+]m which in tum exerts a positive feed-back on the mitochondrial metabolism, thereby ensuring maintenance of the metaboiism-secretion coupling beyond the initiating events.

3. Role of the mitochondria in signal generation

Although an increase in [Ca2+]c is a necessary event in insulin secretion, glucose is aiso capable of stimulating secretion in a manner not involving KATP channels and Ca2+ influx. This is observed at permissive and constant [Ca2+]c [9,10,40]. This effect could be explained by an activation of the mito chondrial metabolism triggered by an increase in [Ca2+]m. We reported that glucose induces [Ca2+]c and [Ca2+]m tises which are associated with the stimulation of insulin secretion [18]. A pivotai role of the mitochondria has also been highlighted by the observations that insulin secretion-deficient diabetes mel litus can be associated with mutations in the mitochondrial genome which encodes several subunits of the respiratory chain complexes [22]. Elimination of the mitochondrial DNA in fi-cell unes after culture with mutagenic compounds results in the complete inhibition of glucose induced insulin secretion. This was associated with impaired oxygen consumption [44] and defective ATP production [20,46]. These studies emphasise the crucial role of mitochondrial metabolism for the glucose mediated [Ca2j~ tise and insulin release and corroborate earlier investigations using mitochondrial poisons or reduced oxygen tension (reviewed in [8]). Transfer of reducing equivaients to the electron transport chain increases the mitochondrial membrane potential (AW~), which enhances the driving force for mitochondrial Ca2+ up take mediated by a low affinity uniporter [13], the threshold of which is approximateiy 300—400 nM [15].

This &Pmdependent CaZ+ entry permits an amplification of [Ca2+]m, relative to [Ca2+]c, further favour ing the stimulation of the Ca2+~sensitive dehydrogenases [18,37]. Stimulatory glucose concentrations have indeed been shown to promote hyperpolarization ofA!P’m in fi-cells [7,12] and in the differentiated fi-cell une INS-1 [24]. As expected, z~!Pmwas depolarised and insensitive to glucose application in ceils deficient in mitochondrial DNA [20].

To characterise the role of mitochondrial activation in metabolism-secretion coupling, MP~, [Ca2+]m and insulin secretion were measured in intact and permeabiiised cells. In order to study the link between


R Maechler and CE. Wollheim / Mitochondria and insulin secretion 257













(b) (c)

.9- 1500



1000 tV

Co n



C o

Fig. 1. Effects of the TCA cycle intermediate succinate (Suc) on [Ca2+]c, [Ca2~]m and insulin secretion in permeabilized TNS-l cells. Celis expressing the Ca2+_sensitive photoprotein aequorin in the cytosol (a) or in the mitochondria (b) were permeabilized with o-toxin and perifused with an intracellular buffer containing 500 nM free Ca2+ and 10 mM ATP The effects of Suc on [Ca2~jm (b) and insulin secretion (c) were measured simultaneously. The efficient clamping of jCa2+]v is demonstrated in panel (a). Reprinted with permission from Maechler et al. [24].

[Ca2+]c, mitochondrial activation and the exocytosis of insulin, we have permeabilised celis with staphy lococcus a-toxin, generating small holes of only 2—3 nm in the plasma membrane and, therefore, largely preserving cellular integrity. This approach tenders possible the clamping of cytosolic [ATP] and [Ca2~]0 at permissive concentrations, as well as the direct stimulation of mitochondrial metabolism by various non-lipophilic substrates. The preparation permits the on-une monitoring of [Ca2+]m and the simultane ous measurement of insulin secretion [24]. As illustrated in Fig. 1(a), the [Ca2+lc could be clamped to the permissive concentration of 500 nM. This was monitored as photons emitted by the Ca2+~sensitive pho toprotein aequorin stably expressed in INS-1 ceils, and stili retained after a-toxin permeabilisation. The tricarboxylic acid (TCA) cycle intermediate succinate transfers reducing equivalents to the electron trans port chain in a


-independent manner on complex II at succinate dehydrogenase. In permeabilised INS-I celis, succinate hyperpolarises the&P’m [24,26]. As aconsequence [Ca2~]m is rapidly increased in a biphasic fashion (Fig. 1(b)). This mitochondrial activation was associated with a marked stimulation of insulin release (Fig. 1(c)). The importance of the [Ca2+]m rise for the secretory response to succinate was demonstrated by the inhibition of secretion when Ca2+ entry into the mitochondria was suppressed with ruthenium red, a blocker of the uniporter [24]. Furthermore, an increase of [Ca~


from 100 to 500 nM markedly enhances succinate oxidation, which underscores the importance of the rise in [Ca2+]m [26].


258 P Maechler ami C.B. Wollheim / Mitochondria and insulin secretion


I insuiin~






Fig. 2. Model for the role of mitochondria in metabolism secretion coupling in Éhe pancreatic j3-cell (sce text for description).

Gicglucose; GAPglyceraldehyde3-phosphate; I ,3DPG I ,3-diphosphoglycerate; Pyrpyruvate; LDHlactate dehydro genase; TCAtricarboxylic acid cycle; red. equ.reducing equivalents; G?glycerophosphate; met-Sucmethyl-succinate;

Leu L-Ieucine; KJC a-ketoisocaproate; A!Pm mitochondrial membrane potential; AW~ plasma membrane potential;

[Ca2~]~cytosolic [Ca2~]; (Ca2~]mmitochondrial [Ca2~]; mt-factormitochondrially-derived factor.

Citrate, another TCA cycle intermediate which does not increase [Ca2+]m in the pemieabilised INS-1 cells, is unable to elicit insulin release unless


Im is raised during its application [24]. fle latter effect can be achieved with a-glycerophosphate which hyperpolarises MPm through activation of com plex II by the FAD-linked glycerophosphate dehydrogenase. It is noteworthy that a-glycerophosphate alone, while raising [Ca2jm, fails to trigger insulin secretion [24]. Taken together, these results suggest that insulin secretion caused by mitochondrial substrates involves the generation of factors which require both a rise0f[Ca2+]m and a supply of carbons for the TCA cycle. The experimental conditions allow the conclusion that the activation of mitochondrial metabolism generates factors other than Ca2+ and ATP, which are capable of inducing insulin exocytosis.

ATP has been considered to be the principal metabolic coupling factor in glucose-induced insulin secretion [27,30]. The overwhelming amount of ATP is generated through oxidative phosphorylation in the mitochondria not only by glucose but also by other metabolisable insulin secretagogues such as leucine, its deamination product a-ketoisocaproic acid, and methyl-succinate [1,25]. The initiating event in stimulus-secretion coupling is the closure ofKATPchannels in the ~-cell plasma membrane, resulting in membrane depolarisation and voltage gated Ca2+ influx [45,47]. In the mitochondria, the generation of reducing equivalents by substrates and their subsequent transfer to the respiratory chain causes hyper- polarisation of the AWm. This permits the rise in [Ca2j~ to concentrations sufficient for the activation of NADH-generating dehydrogenases [28,39]. This feed forward effect of Ca2~ depends on permissive lev els of [Ca2~]c and on the availability of substrates for the TCA cycle, ensuring anaplerotic input [6,35]

(Fig. 2). The enzymatic profiles of the j3-cell are uniquely poised to achieve optimal entry of glucose carbons into the TCA cycle. Thus, escape of carbons in the form of lactate is minimised through low

GP shuttie




R Maechler and C.B. Wollheim IMitochondria and insuhn secrerion 259

lactate dehydrogenase activity [41,42] coupled with high expression of die mitochondrial FAD-linked a-glycerophosphate dehydrogenase which insures reoxidation of cytosolic NADH [5,42]. Moreover, the /3-celI mitochondria display high activity of pyruvate carboxylase, explaining why approximately 50%

of the pyruvate entering the mitochondria is converted to the TCA cycle intermediate oxaloacetate, die remainder being decarboxylated by pyruvate dehydrogenase [23,41].

Insulin secretion can be stimulated with potassium, causing membrane depolarisation, followed by [Ca2j~ and [Ca2+]m rises [18], but potassium does not reproduce the prominent and long-lasting secre tory response to glucose [10,47]. This difference can be explained by the lack of carbon supply to the TCA cycle, as is also the case for a-glycerophosphate in permeabilised cells [24]. We have suggested that an increase in both [Ca2+]m and TCA cycle activity govern die production of the putative coupling factor of mitochondrial origin distinct from ATP and


itself. The messenger molecule could be ~ther one of die TCA cycle intermediates or a derived metabolite. Cytosolic citrate is the precursor of die putative second messenger malonyl-CoA [35]. This pathway is not likely to be implicated, as citrate failed to cause insulin secretion in permeabilised cells [24]. fle nature of the mitochondrial factor, which should cooperate with ATP and Ca2+ to ensure the normal and complete secretory response to glucose, remains to be established.

4. Rote of die mitochondria in the desensitisation of metabotism-secretion coupting

We sought to probe further the existence of the mitochondrial factor by studying desensitisation of insulin secretion. It has been shown that exposure of fl-cells to short successive pulses of glucose resulted in die inhibition of the insulin secretory response to the second stimulus [29]. Desensitisation could occur at any one of die steps of the metabolism-secretion coupling including insulin exocytosis itself [16].

Using INS- I cells stably expressing aequorin in die cytosol or in die mitochondria, we have previously shown diat desensitisation of insulin secretion is associated widi a parallel reduction of the [Ca2j~ and [Ca~]m responses, die latter being more pronounced [18]. When die glucose evoked [Ca2+]m rise is prevented by either a protonophore or an inhibitor of complex I, die second response to glucose (in the absence of inhibitors) is not desensitised [19]. To monitor cytosolic [ATP] in living cells we used an INS I subline expressing high levels of cytosolic luciferase [25]. The cells were exposed to two successive 5 min pulses of 12.8 mM glucose separated by as min recovery period at 2.8 mM glucose. In contrast to the desensitisation of [Ca2+]m and insulin secretion, the 23% increase in [ATP] was the same during bodi stimulation periods [26]. This result is taken as evidence for the existence of die mitochondrial coupling factor generated under conditions of increased [Ca2+]m and distinct from ATP.

In order to investigate the role of the mitochondria in the desensitisation process, glycolysis was by passed by die use of die TCA cycle intennediate succinate. In intact cells, two applications of die celI permeant methyl-ester form of succinate reproduced die results obtained widi glucose, widi respect to insulin secretion, [Ca2+],~ and ATP generation. To study the dissociation between the [Ca2+]m signai and ATP generation, experiments were performed in a-toxin permeabilised INS-l cells which permit the control of the mitochondrial environment in terms of cytosolic [Ca2+] and [ATP]. Under identical conditions as those described in Fig. 1, i.e., 500 nM free Ca2t the [Ca2jm response was completely inhibited during the second exposure to succinate [26]. This inhibitory effect could be reproduced with a-glycerophosphate which also transfers reducing equivalents to complex II of the electron transport chain, in this case originating from die cytosol. The site of desensitisation is not the respiratory chain,


260 P Maechier andC.B.Wo!!heim f Mitochondria and insulin secretion

since succinate was able to induce similar hyperpolarisation of AWm during the two consecutive appli cations. This is in agreement with the absence of desensitisation of the ATP response observed in intact ceils.

The desensitisation of the [Ca2+]m response induced by KCI in intact celis is indirect evidence for the inhibitory effect of Ca2~ on its own handling [18]. KCI (20 mM) evokes increases in [Ca2j~ up to 2 huM. In permeabilised celis, direct applications of Ca2+ in this concentration range clearly caused desensitisation of the [Ca2jm response to the second pulse [26]. Although the molecular nature of the mitochondrial Ca2+ uniporter has not been identified, it appears to have properties similar to those of plasma membrane Ca2+ channels [21]. It could well be that the desensitisation evoked by an increase in [Ca2+]m involves a mechanism similar to that described for L-type Ca2+ channels [48]. In the present discussion the role of Ca2+ as an activator of mitochondrial metabolism has been emphasised. An addi tional function of mitochondria in cellular Ca2t homeostasis has been proposed. Indeed, Ca2+ mobilised from the endoplasmic reticulum was shown to be transferred to the mitochondria [3] and Ca2+ buffering by mitochondria bas been implicated in the regulation of [Ca2+]c microdomains [2].

5. Conclusion

A key role has been attributed to ATP as a coupling factor in the consensus model of nutrient in duced insulin secretion [27,30]. There is accumulating evidence that an increase in the concentrations of Ca2+ and ATP in the cytosol is a necessary though not sufficient condition for an optimal secretory response [10,24,26]. The existence of a mitochondrial factor capable of eliciting insulin exocytosis bas been demonstrated with several experimental paradigms described in this review. The generation of this factor depends on an increase in [Ca2+]m resulting in the activation of the Ca2+_sensitive dehydroge nases in the mitochondrial matrix. However, a [Ca2+]m riseper se is ineffective but when combined with a carbon supply to the TCA cycle, insulin secretion occurs. In cells depleted of mitochondrial DNA, nei ther glucose in intact cells nor succinate in the permeabilised celi preparation elicits insulin release [20].

Furthermore, mitochondrial desensitisation in the fl-cell underlies the desensitisation of the secretory re sponse, despite normal Ai? generation. Under these conditions, the mitochondrial factor is missing due to insufficient elevation of [Ca2jm. Current work aims at defining the mitochondrial factor(s) coupling metabolism to secretion in the fl-cell.


The authors are indebted to Drs T. Pozzan and R. Rizzuto (Department of Biomedical Sciences, Uni versity of Padova) for fruitful collaborations extending over many years. This work was supported by the Swiss National Science Foundation (grant No 32-49755.96). Part of this work was also supported by a European Union Network Grant (tbrough the Swiss Federal Office for Education and Science).


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