Role of mitochondrial calcium in metabolism-secretion coupling in nutrient-stimulated insulin release

Article

Reference

Role of mitochondrial calcium in metabolism-secretion coupling in nutrient-stimulated insulin release

KENNEDY, Eleanor, WOLLHEIM, Claes

Abstract

Glucose-stimulated insulin release from pancreatic beta cells involves a complex series of signalling pathways. In many forms of diabetes, lesions in this process cause or aggravate the diabetic phenotype. A common motif in these cascades is the elevation of intracellular Ca2+

both in the cytosolic compartment ([Ca2+]c) and within the mitochondria ([Ca2+]m). These parameters can be effectively monitored using the photoprotein aequorin which can be targeted to subcellular compartments by transfection. It is shown that physiological concentrations of glucose elicit [Ca2+]c oscillations measured with fura-2, which correlate well with oscillatory NAD(P)H fluorescence in the mitochondria. Aequorin measurements of [Ca2+]m, though unable to detect oscillations on a single cell basis, reveal large increases in intraorganellar [Ca2+] in response to glucose, elevated amino acid levels and depolarizing concentrations of KCI. These oscillations, in turn, mirror changes in the insulin secretion profile. Since several of the key mitochondrial dehydrogenases involved in oxidative phosphorylation are exquisitely sensitive to changes in [...]

KENNEDY, Eleanor, WOLLHEIM, Claes. Role of mitochondrial calcium in metabolism-secretion coupling in nutrient-stimulated insulin release. Diabetes & Metabolism , 1998, vol. 24, no. 1, p.

15-24

PMID : 9534004

Available at:

http://archive-ouverte.unige.ch/unige:35646

Disclaimer: layout of this document may differ from the published version.

1 / 1

Q ^EVIEW

Diabetes & Metabolism (Paris) 1998, 24, 15-24

ROLE 0F MITOCHONDRIAL CALCIUM IN METABOLISM-SECRETION COUPLING IN NUTRIENT-STIMULATED INSULIN RELEASE

E.D. KENNEDY, C.B. WOLLHEIM

SUMMARY - Glucose-stimulated insulin release tram pancreatic ~ cells involves a complex series

q

signali ng pathways. In many forais al diabetes, lesions in this processtause or aggravate the diabetic pheno type. A common motif in these cascades s the elevation of intracellular Ca2 both in the cytosolic compartment ((Ca”J~) and within the mitochon dria ({Ca2’1~). These paramecers can be effeclively monitored usingIhe photoprotein aequorin which can be targeced 10 subcellular compart ments by transfection. Il s shown that physiological concentrations al glucose elicit ICa2’j~ oscillations measured with fura-2, which correlate well with oscillatory NAO(P)H fluorescence in the mitochondrja. Aequo rin measurements of (Ca2’J,,, lhough unable te detect oscillations on single cell basis, reveal large increases in intraorganellar ICa2’J in res panse ta glucose, elevated amine acid levels and depolarizing concen trations cf KCI. These oscillations, in turn, mirror changes in the insulin secretion profile. Since several al the key mitochondrial dehydrogenases involved in oxidative phosphorylation are exquisitely sensitive ta changes in [Ca2’J, it s proposed that alterations in ICa’jm lead ta ncreased acti vity et the tricarboxylic acid cycle and subsequent AI? production.

thereby facilitating exocytosis of insulin tram secretory granules. The involvement of the mitochondria in these processes is examined, as is the putative raIe0fefficient mitochondrial genome transcription and transla tian in normal and diabetic states. Diabetes & Metabolism 7998, 24,

7-16.

Key-words :pancreas, ~ celI, oscillations, calcium, mitochondria,insu lin, secretion.

RÉSUMÉ - Rôle du calcium mitochondrial dans le couplage métaboljsme-secrétion au cours de l’insulinosec,étjon stimulée par les nutriments. La stimulation de linsulinosecrétion par le glucose implique une chaîne complexe de signaux, Dans plusieurs formes de diabète, des anomalies de ce processus causent ou aggravent le phénotype diabéti que. Un élément commun de ces cascades est l’augmentation du Ca”

intracellulaire dans le cytosol QCa2’],) et dans les mitochondries (ICa”J,,). Ces paramètres peuvent être suivis en utilisant l’aequorine, une photoprotéine qui peut être ciblée dans les compartiments subcellulaires par transfection. Des concentrations physiologiques de glucose indui sent des oscillations de Ca”] mesurées grâce au fura-2 qui est bien corrélé à la fluorescence oscillatoire de NAD(P)H dans la mitochondrie.

Les mesures de ICa2’I,,, par l’aequorine, bien qu’incapables de détecter des oscillations au niveau d’une cellule, révèlent de grandes augmenta tions de ICa2’j dans tes organeltes en réponse au glucose, aux acides aminés ou à des concentrations dépolarisantes en KCI. Ces oscillations se superposent aux variations de l’insulinosécrétion, Comme plusieurs déshydrogénases mitochondriales impliquées dans la phosphorylation oxydative sont extrêmement sensibles aux variations de (Ca2’j, il est pro posé que les altérations du lCa2’I,,, conduiraient à une activité accrue du cycle de l’acide tricarboxylique et donc à une production WATP facilitant l’exocytose de l’insuline à partir des granules sécrétoires. (Implication des mitochondries dans ces processus est envisagée, comme l’est te possible rôle du génome mitochondrial dans les états normaux et diabé tiques. Diabetes & Metabolism 1998, 24, 7-16.

Mots-clés: pancréas, cellule p, calcium, mitochondrie,insuhne,secré tion.

C.B. Wollheim, Division de Biochimie Clinique, Dé partement de Médecine Interne, Centre Medical Universitaire, CH- 1211 Geneva 4, Switzerland, TeL: 41-22-702-55-48. Fax.:

41-22-702-55-43,

Received: September 26, 1997.

16 [.0. KENNEDY. C.B. WOLLHEIM Diabetes & Metabolism

Ø

lood glucose homeostasîs is criticallydependent on the Light regulation of insu

I

lin secreLion [rom the pancreatic ~ cdlIII. Insulin is stored in secretory granules of the 3 cell. of which only a small pro portion s released, even following strong stimulation 12].

Glucose and leucine are the two main nutrient stimuli of physiological importance capable of initiating insulin re lease

131.

Their effect is potentiated by a variely of hormones and neurotransmitters, including glucagon-like peptide I (GLP-1), cholecystokinin (CCK) and acetyl choline (ACh) [2, 4].

The consensus model of insulin secretion induced by both glucose and leucine allocates a central role to the mitochondria in metabolism-secretion coupling

13,

5,6]. The pancreatic j3 celI in the rodent expresses predominantly the GLUT-2 glucose transporter which effectively equilibrates the perceived blood glucose concentration across the plasma membrane

[5].

Com pelling evidence indicates that the high Km, glucose phosphorylating enzyme, glucokinase, is the glucose sensor, i.e. the flux-determining reaction of glycolysis

17. 8j.

In fact, one of the subtypes of maturity onset diabetes of the young (MODY) has been associated with mutations in the glucokinase gene resulting in impaired glucose-induced insulin release [8, 9]. Like wise, targeted kr*ck-out of 3-celI glucokinase in mice led to u defective responsiveness to glucose [10]. Fur ther to its conversion to pyruvate, glucose carbons enter the mitoehondria and feed into the tricarboxylic acid (TCA) cycle which generates redueing equiva lents in the form of NADH and FADH2 [3, 5]. These are relayed to the electron transport chain situated predominantly on the matrix side of the inner mito chondrial membrane. Through the increase in respira tion, ATP is generated by the F11F0-ATPase and ex poned w the cytosol [8]. The subsequent increase in che ATP :ADP ratio [Il] promotes membrane depolar ization following closure of the ATP-sensitive K~

(KA.I.p) channels [12]. h has recently been suggested that glucose not only increases ATP concentration but also decreases ADP concentration in ~-TC-3 cells, whieh may be the determining factor in glucose triggered closure of KATP channels [8]. Furthermore, stimulatory glucose concentrations increase the GTP:

GDP ratio by an as yet poorly defined mechanism III .131. Plasma membrane depolarization gates the influx of Ca2 via voltage-sensitive L-type Ca2~ chan nels

1141.

The resultant increase in cytosolic Ca21Ï[Ca29~) displays oscillations which correlate with the glucose evoked rhythmical plasma membrane potential activ ity (151. During bursts of Ca2~ action potentials, spikes of stimulated insulin secretion are observed [16, 171. Moreover, Ca2~ oscillations have been asso ciated with fluctuations in the ATP:ADP ratio, which mosi probably reflect mitochondral activation [18].

This contention is borne out by measurements of os cillatory oxygen consumption that denote a concurrent

change in islet respiration 1191. This review will deal with the role of Ca2 in the coupling of the oxidation of glucose and other nutrient secretagogu~s to the exocytosis of insulin.

• THE ROLE 0F MITOCHONDRIA IN ~-CELL ACTIVATION

Extensive studies have demonstrated a key role for miLochondrial metabolism in nutrient-stimulated insu- lin secretion

15,

6, 20-231. Glucose and leucine oxida tion, as monitored by CO, production by islets, corre lates well with insulin secretion 120, 241. In addition, inhibitors of the respiratory chain and other mitochon drial poisons prevent the initiation of insulin secretion by nutrients [20, 21, 251. In the 3 celI, there is tight coupling between mitochondrial metabolism and gly colysis, the latter being inhibited in response to block ers of oxidative phosphorylation [24, 25]. This distin guishes the

F3

ceil from other tissues which display the Pasteur effect, i.e. stimulated glycolysis caused by mitochondrial blockade 124, 25]. The molecular basis for this has been suggested to be the extremely Iow activity of lactate dehydrogenase (LDH) in the

F~

celI, associated with high levels of mitochondrial, FAD linked glycerophosphate dehydrogenase [25, 26]. The consequences of this enzymatie imbalance are ex ploited by the

13

cell to optimize energy production since the introduction of electrons into the respiratory chain via the glycerophosphate shuttle produces 2 moles of ATP less than that produced by total pyru vate oxidation. The latter is favoured by 10w LDH levels which are indeed the lowest in any mammalian celI type [25. 271. Although there are slight discrepan cies between the LDH values obtained in

I~

cells, which possibly reflect a difference in the cell-sorting procedure, overall activity remains low as compared to other ceIl types, in particular the hepatocyte 125, 27]. As a result of low LDH activity, lactate output during glucose stimulation of sorted

13

cells, but not whole islets, is minimal [20, 25]. This is also observed in INS-l and 13-HC cells, two differentiated cell lines with glucose metabolic profiles similar to those of native

13

cells 125, 28].

It was suggested almost two decades ago by Den ton and colleagues that Ca2 plays a crucial role in the provision of energy during cell activation [29]. They proposed a model in which the rise in [Ca2~]~ was relayed to the mitochondria, causing an increase in the matrix Ca2~ concentration of the organelle ([Ca2~]m).

The mitochondria contain three well-characterized NADH-generating dehydrogenases which are sensitive [30]. These are I) pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl CoA, Ca2~

sensitivity being conferred by a Ca2~-activated phos phatase; 2) NAD isocitrate dehydrogenase, which turns isocitrate into a-ketoglutarate and 3) a-keto glutarate dehydrogenase, which converts a-ketoglu

Vol. 24. ^fl° 1, 1998 MITOCHONDRIAL CALCIUM AND INSULIN RELEASE 17

tarare into succinyl-CoA. The aLLer two enzymes are components of the TCA cycle. In this way, Ca2~ is able 10 facilitate ATP production 10 mee the increased ATP requirenients of ce!! activation (30]. FAD-linkecj cz-glycerophosphate dehydrogenase is aiso Ca2~- activatabie, but this enzyme, owing 10 iLs !oca!ization on the outer surface or the inner mitochondrial mem brane, is sensitive 10 fluctuations in the Ca2~ concen tration in the intermembrane space of the organelle, i.e. those of (Ca2tJc (26,311. In the pancreatic f3 ce!!, aL

!east three of these enzymes have been shown to be Ca2-sensirive 26,

3i-33j.

!n addition, glucose has been shown to activate both PDH

1331

and the glycero phosphate shuttie in intact islets

1341.

The latter was a!so stimu!ated by leucine and ils deamination prod uct, a-ketoisocaproic acid (K1C) (341. !ndeed, remova!

of extracellu!ar Ca2~ has been shown Lo reduce glu cose oxidation but flot total glucose utilization, which argues for a role of mitochondrial Ca2 in signa! gen eration 135,361.

• Ca2-DRIVEN METABOLIC OSCILLATIONS IN THE ~-CELL

Evidence for the rqle of Ca2~ in driving f3-cell metabolism has been gathered from experiments in which (Ca

~(

was measured in parallel with NAD(P)I-I in single, fura-2-loaded f3 cells (37]. In this preparation, stimu!atory glucose concentrations gener ale regular ICa2I~ oscillations in approximately 10%

of cells 1381, with a periodicity of — I .4/min (37].

Likewise, glucose promotes NAD(P)F1 oscillations with a similar periodicity ^(— l.3/min)

1371.

The ICa2~i~. oscillations are reminiscent of those observed in intact mouse and human islets 115-17,39]. In con Lrast, glucose-induced increases in NAD(P)I-I fluores cence were nol resolved inLo oscillations in whole mouse islets

1391.

A possible explanation for this ap parent discrepancy is suggested [rom the resuits shown in Figure I. Whereas 11.2 mM glucose causes large Ca2~ osci!!ations, the corresponding fluctuations in NAD(P)I-I are of comparative!y small amplitude (Fig. lA and B). These latter oscillations could easily escape detection during simultaneous recording from a large number of islet ceils. It has been shown that, though glucose increases NAD(P)I-I fluorescence in isiet ceils, II lowers FAD fluorescence simultaneousiy [22]. This, in conjunction with other biophysical evi dence, strongly favours the notion that the effect of glucose on NAD(P)I-I fluorescence largely reflects in creased NADI-! generation by the mitochondria 122]. It can therefore be inferred that increased (Ca2~], stimu lates mitochondrial NADI-I production.

This hypothesis is strengthened by the abolition of NAD(P)H oscillations in the presence of EGTA, which blocks Ca2~ influx ami glucose-stimulated [Ca29~ rises (Fig. IC and D). Moreover, K depolar

ization promoted increases in both ICa2I~ and Ca2 iniJux-dependent elevations in NAD(P)H fluorescence in single rat

13

^ceils

[37j.

Direct evidence that Ga2 generates NADH in mitochondria was recently oh tained in permeabiized HIT-T15 ceils by a combina Lion of the mitochondrial substrate, pyruvate, with elevated Ca2 concentrations (40(.

To gain further insight into the role of Ca2~ as a regulator of mitochondrial function, ccli unes stably expressing the Ca2~-sensitive photoprotein, aequorin, have been established 1411. These clonai unes were derived from highly differentiated, glucose-sensitive INS- I cells [42]. Glucose-stiinulated insulin secretion profiles remained clearly biphasic and occurred in the physiological concentration range [41]. Stable expres sion of aequorin in the mitochondria is achieved by transfecting cells with a chimera encoding aequorin with an N-terminal targeting sequence comprising the presequence of cytochrome-c oxidase subunit VII!

This INS-! EK-3 cell hne exhibits oscillations in [Ca

~]

when exposed to 10 mM glucose as measured by fura-2, which are similar to those observed in single rat )3 cells (Fig. lE and F). To allow compari son of [Ca2]m fluctuations with corresponding [Ca2~j~ changes when the same Ca2 sensor was used, an INS-I ce!! line stably expressing cytosolic aequorin was also established 1411. In contrasi to more con ventional Ca2 indicators such as fura-2, aequorin nei ther buffers the perceived Ca2~ concentration nor un dergoes any bleaching, which makes it an ideal tool for measuring [Ca2~] in a variety of ceilular compart ments [44].

As shown in Figure 2, increases of glucose in the physiological concentration range between basal 2.8 mM and stimuiatorï 5 mM and 10 mM cause a graded rise in both [Ca

‘i~

and lCa2]~. In other celi types, [Ca2]m has been successfuuly assessed by em ploying aequorin in transieni transfection studies

[43,45,46). This approach can also be applied to both primary rat islet cells (47] and !NS-! celis (Fig. 2B).

The results obtained with transient transfection are qualitatively similar to those seen in the INS- I EtC 3 ce!! line. It shouid be noted, however, that esti mations of basal [Ca2’im after transient transfection with aequorin are subject to variation depending on differences in expression efficiency. This is particu larly pertinent in the monitoring of (Ca2]~ with this method. The celI line stably expressing cytosolic ae quorin overcomes this problem and disp!ays (Ca2j~ in the range 100-150 nM, which is in agreement with values reported for [Ca2~]0 assessed with fluorescent indicators in

13

ceNs and derived ceil lines (Fig. 2C,

[41p.

It is noteworthy that, while resting ICa2Jm and [Ca

~

levels are very close, those observed after stimulation are disparate, being higher in the mito chondria than in the cytosol. This applies not only to glucose as seen in Figure 2 but also to the muscar inic agonist, carbacho!, and the depolarizing agent,

18 E.D. KENNEDY, C.B. WOLLHEIM Diabetes & Metabolism

KCI [4fl. A possible mechanism underlying this am plified response of the mitochondria could be that a proportion of these organelles is organized in micro-

A

domains situated close to L-type Ca2 channels in the plasma membrane and to putative sites of exocytosis.

k has already been demonstrated that small cl~anges in

B

Fia. 1. Effecis 0f increased glucose concentration onfCa2J~ (A and C) and NAD(P)H fluorescence (B and D) in single ratficeils loaded wirh Jura-2.

fCêj~ measurements (nM) and NAD(P)H fluorescence (% basal values) were recorded in parallel experiments using dual excitation microspectro fiuorimetry. Basal glucose concentrations were 4 mM. Figure lE and IF are iwo representative traces cf fCa2I~ measuretnents in Jisra-2-loaded INS-l EX-3 ceils exposed ra increasing concentrations o! glucose. in which basal glucose is 2.8 mM. Reproduced with rite kind pennission of the authors A-D 137] and E amI F (41].

11.2

8.3 8.3 dmMgft

t-,

+

L)n

150 IlS 100

D g

2—

C

P

t-

h

g z II.? mM glc

0 200 400 600 800 1000

limc (s) 400

300 200 100 O

0 200 400 600 800 1000 1200

Time (s) C

400

300 200

100

o

0 00 200 300 400 500 600

Time (s)

E lOmM

5 mM

300

150

25

100

0 100 400 600 800 1000 200

Tirne (s)

F lOmM

5 mM

0 500 1000

lime (s)

o _{500 1000}

Timo (s)

I500

VoL 24, n0 7, 7998

MITOCHONDRIAL CALCIUM AND INSULIN RELEASE 19

800 6%

4%.

200

j1400

1200 [mIe 1600

~600 j400 f200

‘o

350

the extracellular K concentration elicit rises in [Ca2~]m without detectable changes in {Ca2J0 which points 10 a preferential link between Ca2 influx and [Ca2]m elevations [41]. This mechanism is further substantiated by the results shown in Figure 24. The chelation of extracellular Ca2 with an excess of EGTA causes a rapid decrease in [Ca2]m to basal values. As in the case of glucose-stimulated insulin secretion, the increases in both [Ca2]m and ICa2~i~ in celi unes stably expressing aequorin are biphasic.

The influx of Ca2 mb the mitochondria is regu lated by a low-afflnity Ca2 uniporter with a threshold value of approximately 500 nM [48]. Efflux depends

1400 1200 1000

1~~l Gb

s.—.et ______________________

2 SmM EG1A

A

B

I

t,

I

^{—100 soc~d,}

t

f lObTVdGt

—100160554, — ____________________

flnMGb

on Na~/Ca2~ and H~/Ca2~ antiporters [481. Ca2 up take is favoured by hyperpolarization of the mitochon drial membrane potential, which increases the drivittg force for the ion since it is very negative inside (approx. 180 mv). Glucose has been shown to hy perpolarize the mitochondrial membrane potential in both primary

I~

celis and INS-l cells [22~47). The mechanism underlyin~ the amplification of [Ca t]m in comparison with [Ca

~

during glucose stimulation may therefore be two-fold. Firstly, any agent which depolarizes the plasma membrane potential, such as KCI, will cause an increase in [Ca2~}m because of the favourable localization of the.mitochondria. Secondly, nutrient secretagogues, through their stimulation of the respiratory chain and resultant hyperpolarization of the mitochondrial membrane potential, will increase Ca2~ uptake by the organelle.

Dissipation of the mitochondrial membrane poten liaI with the potent protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), caused a lower ing of [Ca2~]~ b approximately 100 nM. This was expected, since the effective collapse of the membrane potential in these conditions results in an equilibration between [Ca29m and [Ca2~]~. In the presence of I CCCP, 10 mM glucose failed 10 elicit any increase in [Ca2tjm (Fig. 3A). This is in agreement with the marked attenuation of the response to 20 mM KCI [41]. k should be noted that, under the same experi mental conditions, KCI-induced [Ca2j~ rises were largely unaffected [49]. The action of CCCP is rapidly reversible since glucose can raise (Ca21m after a re covery period of only 5 min (Fig. 3A). The effeci of CCCP on insulin secretion bas also been studied in rai islets. While the secretory response 10 glucose was significantly but not completely reduced, the corre sponding effects of CCCP on glucose oxidation were only marginally altered, whereas the ATP: ADP ratio remained unchanged [20]. This would indicate a fun damental role for a functional mitochondrial mem brane potential in the generation of metabolic coupling factors other than ATP since mitochondrial glucose metabolism appears unaffected by the mitochondrial poison.

However, when glucose metabolism is severely re stricted by the presence of inhibitors of the respiratory chain, insulin secretion is also reduced, indicating that the two driving forces are equally important [20].

Perifusion of rat islets with amytal, a potent inhibitor of site I of the respiratory chain, in the presence of elevated glucose concentrations led b complete aboli tion of the insulin seeretory profile [211. In addition, as can be seen in Figure 3B, 10 mM glucose fails to elicit an increase in [Ca2]m in the presence of amytal. As expected, the response 10 depolarizing concentrations of KCI is largely unaltered by the inhibition of oxida tive phosphorylation since its ability b increase [Ca2}m is largely independent of the integrity of the mitochondrial respiratory chain. As with CCCP, the C

300 5mMGb

J

²⁵⁰

~ 2%

32

I,2

t,

‘00— - —

— 100 seconds

FIG. 2. Effecis of increasing glucose concentrations on (Ca2j,,, (A and B) and (Ca’)~ (C) in cetis srably apressing aequorin targeted w rite mitochondria (A) or rite cyrosol (C). Panel B represenis typical (Ca2J,, measuren,enrs in !NS-I cells rransiently rransfecred with ae quorin.

20 E.D. KENNEDY, C.B. WOLLHLIM Diabetes & Metabolism

FIG. 3. Effecis of 0m vrai / Cc?’ j,,, measurernenis in glucose.

effects of amytal appear to be complerely reversible.

After comparatively short recovery periods. glucose can again increase [Ca2~]m to levels routinely ob served in the absence of any prior manipulations.

Taken together. these data suggest a close relationship between rises in [Ca2~]m and insulin secretion in re sponse to nutrient. The obvious role of ATP, or per haps more precisely the ATP:ADP ratio, should flot be overlooked. In rat islets, both amytal and antimycin A, another potent inhibitor of respiration, caused severe reductions in the measurable levels of ATP and abol ished glucose-stimulated insulin secretion 120,211. h has been calculated that glucose, to achieve insulin secretion, must increase ATP production above a criti cal threshold [28]. 11 has been suggested that this is due mainly to a mass action effect of glucose, the rate of which is determined by glucokinase, the glucose sensor of the

l~

cell, and ultimately by mitochondrial F11F0-ATPase [8].

It is well-known that agents which interfere with

KATP channel closure by nutrient metabolism inhibit the stimulation of insulin secretion [12]. Diazoxide, the most studied of these agents~ completely inhibits the glucose-mediated rise in [Ca5]m (Fig. 4A). Leu-

cine and its deamination product KIC, which are me tabolized directly by the mitochondria, also depolarize plasma menbrane potential and cause closuze of the

KAq) channels (12,501. Leucine was found to elicit ICa~l0, rises similar to those of glucose (Fig. 4B).

Likewise, ils effects were abolished by the presence of diazoxidc, which again emphasizes the important role of a permissive rise in lCa2~I~ relative to a rise in ICa2Irn since leucine also raises ICa2I~

51,

52].

Fia. 4. Effreis ni cikizoxide (2SOpM) on /Ca”L induced by Me nu (rie,,r sc’crercignç’ues. glucose 10 mM (A) and leucine 10 mM (B).

Work from several laboratories has provided evi dence that glucose is also capable of eliciting insulin secretion in a KATP channel-independent manner (53, 54]. As initially demonstrated by Gembal et al.

1531.

glucose can still cause insulin secretion in the presence of diazoxide, provided that [Ca29~ is raised to a permissive level by membrane depolarization with KCI, thereby allowing an influx of Ca2~ through L-type Ca2 channels. Under these conditions, leucine and D-glyceraldehyde also promote insulin secretion, which indicates that mitochondrially generated cou pling factors are involved in the exocytotic process [55].

lOmM Gic

— 100 seConds A 700

L lpM CCCP lOmM Gic

5600

1500

^__

j

⁴⁰⁰

3300

‘e 200 100

£

B

5000 i—~r~~—_ so~ir~Ô&~s~r

4000

t

3000 E u a 2000

t

5000

—100 seconds

lOrnM GIC2OmM (CI

2000

1500

1000

500

o—100 seconds

(I mM) (A) and CCCP (I pAl) (B) on lAIS-1 EK-3ceNs in response in 10mM

A —. 2500 2501.M Di,zoside

lOtnM Gic 2OreiM (CI IOmM GIc 20’iiM XCI

Ct s,

E

os

tt

sC_u

sE

E

C

B

L 250cM Deazoside IOmM Leucine

5600 —

~500 400

3 300

I,

~o200~

100

C

— Q

—100 seconds lOmM Leucine

VoL 24, n0 1, 1998 MITOCHONDRIAL CALCIUM AND INSULIN RELEASE 21

The generation of such metabolic coupling factors is shown schematically in Figure 5. Pyruvate is formed from glycolysis and enters the mitochondria preferentially due to the Iow LDH levels of the

l~

celi and glucose-responsive celi unes [25,28). In the mito chondria, pyruvate is decarboxylated to acetyl CoA, a reaction catalyzed by lite Ca2~-sensitive enzyme com plex PDH (denoted I in Fig. 5) or carboxylated to oxaloacetate by pyruvate carboxylase [56]. The latter reaction provides anaplerotic input to the TCA cycle [3). Leucine also feeds into the TCA cycle via acetyl CoA production. As previously mentioned, NAD isocitrate dehydrogenase and a-ketoglutarate dehydro genase also constitute Ca2-sensitive reactions (denoted 2 and 3 respectively in Fig. 5) [30]. Through the pro duction of reducing equivalents, electrons are fed into the respiratory chain and ATP is synthesized. Glucose metabolism also stimulates the Œ-glycerophosphate (GP) shuttle [34]. This is one of the shuttles which transports reducing equivalents from the cytosol to the mitochondria. Its mitochondrial component, the FAD linked GP dehydrogenase, responds to fluctuations of (Ca2~], in view of its location on the outer surface of the inner mitochondrial membrane [26, 31]. The nutri ent secretagogues hyperpolarize the mitochondrial membrane potential through increased activity of the respiratory chain 122, 4*]. As a result of stimulated mitochondrial metabolism, the cytosolic ATP:ADP ra tio increases, which is instrumental in the closure of KATP channels (Il, 121. The resultant depolarization and electrical activiry promote gated Ca2 influx and elevation of [Ca2j~ 121.

The actions of the cytosolic Ca2 signal are two fold:

1) it triggers exocytosis of the insulin-containihg secretory granules, a process also requiring ATP and GTP [2,13] and

2) it raises [Ca2]m, which is favoured by the afore mentioned hyperpolarization of the organellar mem brane potential.

At the present time, k cannot be definitively estab lished which of the two actions is more dominant in the KATP channel-independent stimulation of insulin secretion by nutrients. There is now evidence to sug gest that the increase in [Ca~]m stimulates the gene ration of novel mitochondrially derived coupling fac- tors capable of promoting insulin exocytosis when [Ca2~]0 is clamped to permissive levels (< I MM) in the presence of saturating ATP concentrations [47].

These conditions were imposed in INS-1 cells perme abilized with Staphylococcus a-toxin which creates small holes solely in the plasma membrane (57]. Only agents such as succinate which are capable of both fueling carbons into the TCA cycle and raising [Ca21m, were found to induce exocytosis of insulin.

Accordingly, a-glycerophosphate, which hyperpolar izes the mitochondrial membrane potential and causes a marked increase in [Ca2]m, failed to enhance insu- lin secretion. Although the nature of the putative mi tochondrial coupling factor(s) remains to be defined, it would appear most likely that products of the TCA cycle or compounds synthesized downstream thereof mediate the secretory effect. Acyl CoAs such as malo nyl CoA and palmitoyl CoA have been proposed to be

Sic ^~. GAP—*-I3DPG^—*-—.ø-Pyr

-f,.-

^lactate

~LDH

_______ aGP

__

t

^—

GP shuttie

j

DHAP

Fio. 5. Schenwric represenlazion of Me nutrienr-sgimuiaved signalling-secresion paMways in Me pancrearicficeiL

22 BD. KENNEDV, C.B. WOLLHEIM Diabetes & Metaboliem

coupling factors during nutrient stimulation of insulin secretion

31.

Since the latter opens rather than closes

KATP channels, the role of these Iipid derivatives re mains unclear 1581 A relatively small effect of glucose on insulin secretion is also observed when both pro tein kinases A and C are stimulated under conditions in which [Ca2~]c remajns aL basal levels

[591.

Whether this response involves the same coupling factor(s) as the Ca2~dependent metabolism-secretion process is as yet unknown.

• p-CELL MITOCHONDRIAL FUNCTION AND IMPAIRED INSULIN SECRETION The key role of mitochondrial metabolism in nutrient-stimulated insulin secretion is further high lighted by the well-known capacity of methylsucci nate, the cell-permeant analogue of succinate, to mimic the eftècts of glucose and leucine [60]. The GK rat, a model of non-insulin-dependent diabetes melli Lus (NIDDM), displays impaired glucose-stimulated insulin secretion. lnterestingly, the secretion stimu lated by methylsuccinate was also deficient in isolated GK rat islets, whereas activation of exocytosis follow ing depolarizatio,n with K was normal [61]. The mo lecular basis of ibis defect is poorly understood. The etiology of another diabetic syndrome in an animal model, the BDEtDB rat, has been linked to a mutation in subunit 6 of F11F0-ATPase

[62].

This mitochondri aIly encoded protein determines intraorganellar ATP production. The islets from these animais only display j mpaired glucose-stimulated insulin secretion after culture at high glucose concentrations [81. This is perhaps to be expected from a single point mutation in a protein which is transcribed and translated in the mitochondria. More severe phenotypes would be cx pected from a mutation affecting translation in a more global manner. Paramitochondrial DNA (mtDNA) is a highly compacted, maternally encoded circular DNA of 16,569 bp, present in several copies within each mitochondrion [631. MtDNA undergoes comparatively rapid replication during which deletions and mutations are less efficiently repaired than those encountered in the nuclear genome. A number of diseases have now been associated with such alterations in mtDNA, in cluding myoclonic epilepsy and ragged red fibre dis ease (MERFF), Leber’s hereditary optic neuropathy (LFION) and mitochondrial encephalomyopathy, iactic acidosis and stroke-like syndromes (MELAS) [64]. Pa tients with MELAS are also often diabetic [65]. Simi larly, another newly-defined subtype of maternally in herited diabetes often accompanied by deafness (MIDD) is caused by a mutation in tRNA(L~~R) at position 3243 of mtDNA [66]. Since this mutation affects the translationai efficiency of mtDNA, it may be expected to have more severe consequences than isolated point mutations in the mtDNA-encoded sub units of respiratory chain enzymes. In keeping with

this idea, the same tRNA(~M~) mutation is ob served in patients affected by the disabling neuromus cular disease, MELAS. Defective glucose-Stimulated insulin secretion appears to be the crucial feature un derlying this diabetic syndrome which, however, ofien evolves into insulin-dependent diabetes melhtus 65,66j. Mutations in mtDNA may, however, also be implicated in more common forms of NIDDM since a report from Japan has shown that 5 % of NIDDM patients exhibit a point mutation at position 3394 of mtDNA in the mitochondrially-encoded NADH dehy drogenase subunit 1167].

Recently, the importance of mtDNA in the develop ment of this pathophysiological entity has been eluci dated in studies on the insulin-secreting cell une MIN-6. Following chemical ehmination of mtDNA, glucose-stimulated insulin secretion was completely abolished. The pivotai role of the mitochondria in this process was underscored by the restoration of the secretory response through replenishment of mtDNA deficient cells with functional mitochondria using cy brid fusion techniques [68]. In similarstudies in INS-i cells depleted of mtDNA, we found that insulin re lease was abrogated in the presence of glucose, whiie the response to K depolarization was unaffected [69].

• PERSPECTIVES

Converging evidence points strongiy to the funda mental importance of ICa2~], in both the onset and sustained secretory response to glucose in

F~

ceils.

Initiation of Ca2~ influx following glucose metabolism wiII assure the continued generation of coupling fac- Lors by the mitochondria. As [Ca2]0 oscillates in re sponse to nutrient stimuli via osciiiiating membrane potential, it is most likely that these events are relayed to the mitochondria in the form of [Ca2~]m oscilla tions. Such [Ca2]m oscillations would lead to meta bouc fluctuations, as demonstrated by changes in NAD(P)H fluorescence in single f3 cells [37]. How ever, aL the present time, the [Ca2~]m signal recorded in ceil lines stabiy expressing aequorin in the mito chondria cannot be resolved into single transient spikes in individual ceils. The reported measurements were obtained from populations asynchronously re sponding to the stimuli. To achieve single celi record ings, higher expression levels of aequorin must be obtained. To this end, microinjection techniques have been used to introduce aequorin cDNA directiy into the nuclei of CHO cells [70]. Furihermore, it wiill be important to characterize the nature of the mitochondrially-derived coupling factors generated in a Ca2-dependent fashion during nutrient stimulation of insulin secretion. In particular, it wiili be essential to delineate whether the Ca2*~sensitive step is indeed one of the Ca2~-activated mitochondrial NADH-generating dehydrogenases or a hitherto iess weli-deflned meta

Vol. 24, n° 1, 1998

MITOCHONDRIAL CALCIUM AND INSULIN RELEASE 23

bouc reaction. The characterjzatjon of these Ca2~- dependenifunctions in die t3-ceut should, in turn, help elucidate the impairment of insulin secretion in several subtypes of NIDDM.

Acknov4edgnienrs—This worl< was supporied by the Swiss National Science Foundation (No. 32-32376.91) the Carlos and Elsie de Reuter Foundacion for Medical Research (Geneva) and the Karl Mayer Stiftung. The work was also supported in large

partby a European Union Network Orant to T. Pozzan and C.B.

Wollheim (through the Swiss Federal Office for Education and Science). The authors also wish to thank Dr. Pierre Maechler for helpful discussion.

REFERENCES

Porte O ir. ~‘celIs in type Il diabetes mellitus (Banting Lecture 1990). Diaberes, 1991. 40, 166-180.

Wollhcim CB. Lang J. Regazzi R.,The exocycotic process of insulin secretion and lis regulation by Ca” and O~protcins. Diabetes Rev

1996. 4, 276-297.

3 Prentki M. New insightsmb pancrcaiic ~-cell metabolic signaling in insulin secretion, Eur J Endocrjnoj, 1996. 134, 272-286.

4 Thorens B, Waeber G. Glucagon-like peptide.l and the control of insulin secretion in the normal siaic and in NIDDM, Diabetes. 1993, 42. 1219-1225.

5 Newgard CB. Mcoany JO. Melabolic coupling faclors in pancrcalic 3-celI signal transduction, fnnu Rei’ Biochen,. 1995, 64. 689-7 19.

6 Gcrbitz KO, Gempel K. Brdiczlca D. Gcnetic, biochemical and clini.

cal implications cf the ccllular cncrgy circuit. Diabetes. 1996, 45.

113-126.

7 lynedjian PB. Mammalian’glucokinase and ils gene. Review anicle, BiochemJ, 1993. 293, l-13.

8 Matschinsky FM. A lesson in mciaholic rcgulation inspired by the glucokinase glucose sensor paradigm (Banting Lecture 1995). Dia betes, 1996, 45, 223-241,

9 Froguel PH, Zouali H. Vionnet N et o? l-amilial hypcrglycemia duc o mutations in glucokinase dehnpiion of a subtype of diabetes mellitus, N Eng? J MeS. 1993. 328, 697 702.

10 Terauchi Y. Sakura H. Yasuda K et o? Pancrcatic 3-celI-specific targcted disruption of glucokinase gcne J Bio? Chen.. 1995, 270, 30253 -30256.

Il Detimary P, Van den Berghe G. Henquin JC. Concentration depen dcncc and time course cf the efl’ects of glucose on adenine and guanine nucleotides in mouse pancrcatic islcts. J Bio? Client, 1996.

27). 20559.20565.

12 Ashcroft FM, Rorsman P. Elcctrophysiology of the pancreatic r3.cell.

Prog Biophys Molec Bio?. 1989. 54. 87 143.

13 Meredith M. Rabaglia ME, Met,. SA. Evidcncc or a oie for GTP in the potentiation of Cr’.induccd insulin secretion by glucose in intact rat isiets, J Clin !nvesI, 1995, 96. 811-821.

14 Ashcroft FM, Proks P, Smitls PA, Àmmt,l~ C, Bokvisi K, Rorsman P.

Stimulus-secretion coupling in pancreattc 3’cell. J Ce?? Biochem, 1994, 55(Supp?.), 54-65.

15 Santos RM, Rosarjo LM, Nadal A, Garcia-Sancho J, Soda B, Valdeolinillos M. Widespread synchronous lCa2’l, oscillations due to bursting electrical activiiy in single pancreatic islets. Pflùgers Arch. 1991, 418, 417-422.

16 Gilon P, Shepherd RM, Henquin JC. Oscillations of secretion driven by oscillations of cytoplasmic Ca2 as evidenced in single pancreatic islets. J Bio? Client, 1993. 268, 22265.22268.

17 Hellman B, Gylfe E, Bergsten P e’ al. Glucose induces oscillatoiy Ca2 signalling and insulin release in human pancreatic 3-celis, Diabetologia, 1994,37 (suppl. 2), SI l-820.

18 Nilsson T, Schuliz V, Berggren P.O. Corkey BE, Toraheim K. Tem poral pattems of chanjes in ATP/AOP ratio, glucose 6-phosphate and cytoplasmic free Ca2 in glucose-stimulated pancreatic 3-ceils, Bio- client J, 1996, 314, 91-94.

19 Longe EA, Tornhein, K, Deeney JT et al. Oscillations in cytosolic free Ca1, oxygen coqsumption, anti insulin secretion in glucose stimulaLed rat pancreatic islets. J Bio? Client, 1991, 266. 93I4.9~I8.

20 Malaisse WJ, Hutton JC, Kawazu S, Flerchuelz A, Valverde I, Sener A. The stimulus secretion coupling of glucose-induced insulin re lease, XXXV. The links between metabolic and cationic events.

Diabetologia. 979. 16, 331.341.

21 Ohta M, Nelson D, Nelson J, Meglasson MD, Erecinska M. Oxygen and temperature dependence of stimulated insulin secretion in ko iated rat islets of Langerhans, J Bio? Chem, 1990, 265, 17525.17532 22 Duchen MR, Smith PA, Ashcroft FM. Substrate-dependent changes in mitocliondrial function, intracellular free calcium concentration and membrane channels in pancreatic 3’ceils. Biochem J. 1993. 294.

35-42.

23 Malaisse Wi, Sener A, Malaisse.Lagae F et al. The stimulus secretion coupling of amino acid’induced insulin release. Metabolic response of pancreatic islets to L-glutamine and L-leucine. J Bio?

Client. 1982, 257, 8731-8737,

24 Hellman B, Idahl LA, Sehlin J, T~ljedal lB. Influence cf anoxia on glucose metabolism in pancreatic islets: lack of correlation between fn,close- I .6-diphosphate and apparent glycolytic flux. Diabetologia, 1975, 11. 495-500,

25 Sekine N, Cirulli V, Regazzi R e, al. Low lactate dehydrogenase and high mitochondnal glycerol phosphate dehydrogenase in pancreatic 3-ceils, J Bio? Client, 1994, 269, 4895-4902.

26 MacDonald Mi, Brown Li. Calcium activation of mitochondrial glycerol phosphate dehydrogenase restudied. Arch Bioclien, Biophs’s,

1996, 326, 79-84.

27 Jijakli H, Rasschaen J. Nadi AB, Leclercq-Meyer V, Sener A. Mai aisse WJ, Relevance of lactate dehydrogenase activity to thc control or oxidative glycolysis in pancreatic islet J3’eells. .4rcli Bioc’hem Biophys. 1996, 327, 260-264.

28 Liang Y, Bai G, Doliba N et al. Glucose metaboIism and insulin release in mouse pHC9 cells as model for wild.type pancrcatic 3-cells. An. J Physio?, 1996, 279 (Endocrinol Metab 33): E846- E857.

29 Denton RM, MeCon’nack JG. On the role of the calcium transport cycle in heart anti other mammalian mitochondria. FEBS Leu, 980.

119, I-8.

30 McCormaek JG, Halestrap AP, Denion RM. Role of calcium ions in regulation of mammalian intramitochondrial metaboIism. Phvsiol Ber, 1990, 70, 391-425,

31 Rutter GA, Pralong WF, Wollheim CB. Regulation of mitochondrial glycerol-phosphate dehydrogenase by Ca2 within electropermeabi lized insulin secreting cells (INS.l). Biochim Biophys Acta. 1992, 1175, 107-113,

32 Sener A, Rasschaert J, Malaisse WJ. Hexose metabolism in pancrc atie islets, Participation of Ca2-sensitive 2-ketoglutarate dehydroge nase in the regulation of mitochondrial function, Biochim Biophys Acta, 1990, 1019, 42-50.

33 McCormack .10, Longo EA. Corkey BE. Glucose-induced action of pyruvate dehydrogenase in isolated rat pancreatic islets. Biocie,n J.

1990, 267, 527-530.

34 Sener A, Malaisse WJ, Hexose metabolism in pancreatic islets.

Ca2tdependent activation of the glyeerol phosphate shuttle by nu- trient secretagogues. J Bio? Client, 1992, 267, 13251.13256.

35 Sener A, Malaisse WJ. Hexose metabolisni in pancreatic islets.

Regulation of D.{6-l4C~glucose oxidation by non-nutrient secreta gogues. Mol Ce?? Endocrinol. 1991, 76, l’6.

36 Malaisse WJ, Glucose-sensing by the pancreatic 3-celi : the mito ehondrial pari. miJ Bioclien,, 1992, 24. 693-701,

37 Pralong WF, Spât A, Wollheim C8. Dynamic paeing of cdl metabo.

I,sm by intracellular Ca2 transients. J Bio? Client. 1994, 269, 27310.

27314.

38 Theler JM, Mollard P. Guérineau N et aL Video imaging of cytosolic Ca2 in pancreatic 3-celis stimulated by glucose, carbachol, and ATP.

J Bio? Client, 1992, 267, 18110-18117.

39 Gilon P, Henquin JC. Influence of membrane potential changes on cytoplasmic Ca2 concentration in an electrically excitable ccli, the insulin-secreting pancreatic 3.ceIl. J Bio! Client, 1992. 267, 20713- 207 20.

2

24 E.D. KENNEDV, GB. WOLLHEIM Diabates & Metabolism

40 Civalek VN, Decney iT, Shalosky Ni e’o?. Regulation of pancreatic

~-ceII mitochondrial metabolism inouence of Ca2’, substrate and AOl’. Bioc’he,n J, 1996. 318, 615-621.

41 Kennedy ED, Rizzuto R, Theler 1M et al. Glucose-stimulated insulin secrelion correlates wilh changes in miccchondrial and cytosclic Ca’ in acquorin cxpressing INS-i ceils, J Clin loves:, 1996, 98, 2524.2538.

42 Asfari M, Janjic D, Meda P, Li G, Halban PA, Wciiheim CB, Establishment cf 2-mercaptoethanol dependeni differenliated insulin secreting ccli unes. Endocrino?og% 1992, 130. 167-178.

43 Rizzuto R, Simpson AWM, Brini M. Pczzan T. Rapid changes of mitochondijai Cr revealed by specifically targeted recombinant aequorin. Nature, 1992, 358, 325-327.

44 Rizzuto R, Brini M, Pozzan T. Targeting recombinant aequorin 10

specihc intracellular organelles Mciii. Ce!? Bio?.. 1994, 40, 339-358.

45 Brandenburger Y, Kennedy ED, Python CP e? al. Possible rob for mitochondrial calcium in angiotensin Il- and potassium-stimulated steroidogenesis in bovine adrenal glomerulosa ceils. Endocrinology,

1996. 137 55445551

46 Lawrie AM, Rizzuio R, Pozzan T, Simpson AWN. A robe for calcium influx in thc regulation cf mitochondrial calcium in endothelial colis.

J Bio! Chen,. 1996, 271, 10753-10759.

47 Maechler P, Kennedy ED, Pozzan T, Wollheim CB. Mitochondrial activation directly triggers the exocytosis cf insulin in permeabilized pancreatic ~-cells. EMBO J, 1997. 16, 3833-3841.

48 Gunter TE, Gunter KK, Sheu SS, Gavin CE. Mitochond,ial calcium transport physiological and pathological relevance. 4m J Physio?, 1994. 267 (CeIl Physiol. 36) C313-C339.

49 Rutter GA. Theler JM, Murgia M, Wollheim CB, Pozzan T, Rizzutc R. Stimulaied Ca2’ influx taises miiochondrial free Ca’ to suprami cromolar lcvels in a pancreatic 13-ceIl une. Possible role in glucose and agonisi-induced insulin secretion. J Bio? Chem, 1993, 268, 22385-22390.

50 Ashcroft FM, Ashcrofi SiH, Harrison DE. Effects cf 2-ketoisocaproate on insulin release and single potassium channel activity in dispers~d rat pancreatic ~-ceIIs. J. PhysioL, 1987, 385, 5 17-529.

SI Gylfe E. Nutrieni secretagogues induce bimodal early changes in cytoplasmic calcium of insulin-releasing ob/ob mouse 3-ceils. J Bio?

Chem, 1988. 263. 13750-13754.

52 Weinhaus Ai, Poronnik P, Cook DI. Tuch BE. Insulin secretagogues, but n°c glucose, stimulate an increase in [Ca211 in the fetal rat ~-cell.

Diabejes, 1995. 44, I I 8 124

53 Gembal M, Gilon P, Henquin iC. Evidence that glucose can control insulin release independently from ils action on ATP-sensitive K’

channels in mouse 3-colIs. J Clin Invesi, 1992, 89, 1288-1295.

54 Sato Y, Aizawa T. Komatsu M, Okada N, Yamada T. Dual functional role cf membrane depolarizationlCa2’ influx in rat pancreatic 13-cell.

Diabezes, 1992. 4!, 438-443.

55 Gembal M, Delimary P, Gilon P, Gao ZY, Henquin JC. Mechanisms by which glucose can control insulin release independenlly from its action on adenosine triphosphate-sentitive K channels in mense 3-cells. J C?in mues:, 1993, 91, 871-880. à

56 MacDonaid Mi. Feasibiliiy of a mitochondrial pyruvate malate shuttlc in pancreatic islets. Furiher implication cf cytosolic NAD(P)H in insulin secrelion. J Bio! Chem, 1995, 270, 20051- 20058.

57 ionas iC. Li G, Palmer M. Waller U, Wollheim CB. Dynamics cf Ca’ and guanosine S’ly-thio]triphosphale action on insulin secte tion from a-toxin-permeabilized HIT-Tl5 cells. Biochem J, 1994, 301, 523-529.

58 Larsson O, Deeney .10, Branstrom R. Berggren P-O. Corkey BE.

Activation of the ATP-sensiiive K’ channel by long chain acyl-CoA.

J. Bio?. Chem., 1996, 271. 10623-10626.

59 Komatsu M, Schennerhon, T, Aizawa T, Sharp GWG: Glucose stimulation of insulin release in the absence cf exiracellular Ca” and in the absence of any increase in intracellular Ca” in rat pancreatic islets. Proc Nazi ,4cad Sel, 1995. 92, 10728-10732.

MacDonald Mi, Fahien L. Glyceraldehyde phosphate and methyl esters cf succinic acid. Diabetes, 1988, 37, 997-999.

Katayama N, Hughes Si. Persaud SI, Joncs PM, Hcwell SL. Insulin secretion from islets of GK rats is n°1 impaired after energy gener ating steps. Mo? Ce!! Endocrinol, 1995, 111, 125-128.

62 Mathews CE, McGraw RA, Berdanier CD. A point mutation in the mitochondrial DNA cf diabetes-prone BHEIcdb rats. FASEB J, 1995, 9, 1638-1642.

Wallace DC. Diseases cf the mitochondrial DNA. Annu R~ Bio chem, 1992,61, 1175-1212.

iohns DR. The other human genome DNA and disease. Nature Mcd, 1996,2, 1065-1068.

65 Kadowaki T, Kadowaki H, Mon Y e? o?. A subtype cf diabetes melliius associated with a mutation cf mitochondrial DNA. N Eng? J Mcd. 1994, 330, 962-968.

66 Maassen iA, Kadowaki T. Matenially inhenited diabetes and deaf ness: a new diabetes subtype. Diabeio?ogia, I 996, 39, 375-382.

67 Hirai M. Suzuki S, Onoda M et aL Mitochondrial DNA 3394 muta tion in Ihe NADH dehydrogenase subunit I associated wiih non insulin-dependeni diabetes mellitus. Biochem Biophys 8es Commun, 1996, 219, 951-955.

68 Soejirna A, moue K, Takai D e? ai. Mitochondrial DNA is required for regulation cf glucose-stimulaied insulin secrelion in a mouse pancreatic 3-cell line, MIN6. J Bio? Chem, 1996, 271, 26194-26199.

69 Kenncdy ED, Maechler P, Wollheim CB. Eflects of depletion cf mitochondrial DNA on metabolism-secretion coupling in INS-1 colis. Diabe:es, 1998. in press.

70 Ruiter GA, Bumett P, Riizuto R et aL Subcellular imaging cf intramitocliendrial Ca” with recombinant targeled aequorin Sig niheance for the regulation cf pyruvate dehydrogenase activity. Proc Na?? Acad Set. 1996, 93, 5489-5494.

60 61

63 64

C Maison, Paris, 1998