Cell Metabolism Resource

Mitochondrial Matrix Calcium Is an Activating Signal for Secretion

Andreas Wiederkehr,1,2,* Gergo} Szanda,3 Dmitry Akhmedov,1 Chikage Mataki,4 Claus W. Heizmann,5 Kristina Schoonjans,4 Tullio Pozzan,6 Andra´ s Spa¨ t,3,7 and Claes B. Wollheim1,* 1Department of Cell Physiology and Metabolism, University of Geneva, University Medical Center, 1 Rue Michel-Servet, 1211 Geneva 4, Switzerland 2Alberta Diabetes Institute, University of Alberta, Edmonton, Canada T6G 2E1 3Department of Physiology, Semmelweis University, H-1444 Budapest, Hungary 4Laboratory of Integrative Systems and Physiology, Ecole Polytechnique Fe´ de´ rale de Lausanne, 1015 Lausanne, Switzerland 5Department of Pediatrics, University of Zurich, 8032 Zurich, Switzerland 6Department of Biomedical Sciences, Neuroscience Institute of Italian CNR and Venetian Institute of Molecular Medicine, 35121 Padova, Italy 7Laboratory of Neurobiochemistry and Molecular Physiology, Hungarian Academy of Sciences, H-1444 Budapest, Hungary *Correspondence: [email protected] (A.W.), [email protected] (C.B.W.) DOI 10.1016/j.cmet.2011.03.015

SUMMARY The organelle is able to accumulate large amounts of Ca2+, but only a minute fraction is in the free, unbound form (Chalmers and Nich- Mitochondrial Ca2+ signals have been proposed to olls, 2003; McCormack et al., 1990). This free Ca2+ concentration is accelerate oxidative metabolism and ATP production tightly regulated (Gunter and Sheu, 2009). Under resting condi- to match Ca2+-activated energy-consuming pro- tions, the Ca2+ uptake and release mechanism ensures that the 2+ 2+ cesses. Efforts to understand the signaling role of mitochondrial Ca ([Ca ]m) is maintained in the range of 100– 2+ 2+ mitochondrial Ca2+ have been hampered by the 300nM, similar to the cytosolic Ca concentration ([Ca ]c;100nM) inability to manipulate matrix Ca2+ without directly (Babcock et al., 1997; Kennedy et al., 1996; Rizzuto et al., 1994). 2+ Many , neurotransmitters, and nutrients induce cyto- altering cytosolic Ca . We were able to selectively 2+ 2+ 2+ solic Ca transients as a consequence of mobilization of Ca buffer mitochondrial Ca rises by targeting the 2+ 2+ from the endoplasmic reticulum (ER) and/or Ca influx from Ca -binding S100G to the matrix. We find the extracellular space. Through the efficient uptake of Ca2+, 2+ that matrix Ca controls signal-dependent NAD(P)H via a partially identified uniporter (Perocchi et al., 2010), mito- 2+ formation, respiration, and ATP changes in intact chondria shape these [Ca ]c rises and restrict the spreading of cells. Furthermore, we demonstrate that matrix Ca2+ Ca2+ from microdomains (Babcock et al., 1997; Rizzuto et al., increases are necessary for the amplification of 1993; Rizzuto and Pozzan, 2006; Szanda et al., 2006). During sustained glucose-dependent insulin secretion in such stimulation mitochondrial Ca2+ uptake exceeds the 2+ b cells. Through the regulation of NAD(P)H in adrenal capacity of the mitochondrial Ca export systems. As a conse- 2+ m glomerulosa cells, matrix Ca2+ also acts as a positive quence, [Ca ]m transiently increases from 0.5 to several M signal in reductive biosynthesis, which stimulates (Babcock et al., 1997; Kennedy et al., 1996; Rizzuto et al., 1994). Based on the stimulation of several matrix dehydrogenases by aldosterone secretion. Our dissection of cytosolic Ca2+, it has been proposed that one of the main roles of [Ca2+] and mitochondrial Ca2+ signals reveals the physiolog- m 2+ is to serve as an activating signal for oxidative metabolism (Mc- ical importance of matrix Ca in energy metabolism Cormack et al., 1990). Ca2+-mediated activation of dehydroge- required for signal-dependent hormone secretion. nases will provide more reducing equivalents, thereby acceler- ating mitochondrial respiration and ATP synthesis (Balaban, 2009; McCormack et al., 1990). Consistent with such a regulatory INTRODUCTION function Ca2+ transients display a very close kinetic correlation with NAD(P)H increases (Duchen, 1992; Hajno´ czky et al., 1995; The main metabolic function of mitochondria is to oxidize Luciani et al., 2006; Pralong et al., 1992, 1994). Furthermore, 2+ substrates generating reducing equivalents, which serve as clamping the [Ca ]c at a low concentration or preventing stim- 2+ a source of electrons for the respiratory chain. Electron transport ulus-dependent Ca signals inhibits CO2 generation and mito- is linked to proton extrusion to form an electrochemical gradient chondrial ATP production (Hellman et al., 1974; Jouaville et al., across the inner mitochondrial membrane. This gradient is the 1999; Kennedy et al., 1999; Maechler et al., 1998). Ruthenium driving force for the ATP synthase which, depending on the red, an inhibitor of mitochondrial Ca2+ uptake, prevented the tissue studied, is responsible for a very large proportion of total Ca2+-induced increase in ATP formation by suspended heart cellular ATP synthesis (reviewed in Duchen, 2004). Mitochondria mitochondria (Territo et al., 2000) as well as Ca2+-induced aldo- also accomplish other cell-specific functions including steroido- sterone production in permeabilized glomerulosa cells (Capponi genesis (Spa¨ t and Hunyady, 2004). et al., 1988); however, this drug and the related compound Mitochondria are key players in cellular Ca2+ signaling (Duchen, Ru360 have several other effects (Hajno´ czky et al., 2006 and 2004; Rizzuto and Pozzan, 2006; Wiederkehr and Wollheim, 2008). references therein).

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The direct demonstration of the role of mitochondrial matrix more, neither the wild-type nor the mitochondrially targeted Ca2+ in intact cells has not yet been achieved. In particular, the S100A1 (mitoS100A1) altered the glucose-stimulated ATP 2+ 2+ relative contribution of [Ca ]c and [Ca ]m in cell activation response (data not shown). remains to be established. Steroidogenesis illustrates well this gap in knowledge as a role has been attributed to both cytosolic MitoS100G Specifically Buffers Mitochondrial Matrix and mitochondrial Ca2+ in mediating the action of stimulatory Ca2+ Rises agents on aldosterone secretion (Cherradi and Capponi, 1998; To assess the effect of mitoS100G on mitochondrial Ca2+ Spa¨ t and Hunyady, 2004). signals, we first studied HeLa cells (Figures 2A–2C). Histamine In this study, we present a molecular biological approach that mobilizes Ca2+ from the ER leading to a rapid [Ca2+] transient. allows us to specifically buffer mitochondrial, but not cytosolic, m Expression of mitoS100G markedly lowered the average ampli- Ca2+ signals. Attenuation of mitochondrial Ca2+ transients tude of the transient (À57%) when compared to control infected reduces stimulus-dependent NAD(P)H formation, mitochondrial cells without induction of the transgene (n = 5; p = 0.0004; Fig- respiration, and ATP synthesis. We further demonstrate a control 2+ ure 2A). DOX alone or expression of mitoECFP did not alter the function for mitochondrial Ca in metabolism-driven cellular 2+ [Ca ]m response (Figure 2C and data not shown). Expression functions such as insulin and aldosterone secretion. 2+ of mitoS100G had no impact on the [Ca ]c rise induced by hista- mine (Figure 2B). RESULTS 2+ 2+ MitoS100G also reduced [Ca ]m increases after influx of Ca from the extracellular space, studied in INS-1E cells, which are Mitochondrial Targeting of S100G electrically excitable. Depolarization with 30 mM K+ opens The role of mitochondrial Ca2+ as a signal was assessed by buff- voltage-gated Ca2+ channels, raising [Ca2+] to a peak value of ering mitochondrial matrix Ca2+ rises. This was accomplished c about 1 mM(Figure 2E). The basal [Ca2+] and the amplitude of through the expression in the mitochondrial matrix space of c the Ca2+ response to plasma membrane depolarization were the Ca2+-binding protein S100G (K of 300-500nM; previously d indistinguishable whether or not mitoS100G was expressed (Fig- named D-9k; Linse et al., 1991). S100G belongs to ure 2E). In contrast, the mitochondrial Ca2+ rise in response to the large family with a canonical as well as 30 mM K+ was strongly reduced (À45%; Figure 2D). The results a S100-specific Ca2+ binding EF hand (Marenholz et al., 2004). demonstrate that matrix-targeted mitoS100G is capable of S100G is a monomeric [Ca2+] buffering protein of 9 kDa. For c specifically buffering [Ca2+] rises. efficient mitochondrial delivery of S100G a tandem repeat m coding for the mitochondrial targeting sequence of cytochrome c oxidase subunit 8 was fused in frame in front of the coding Mitochondrial Ca2+ Acts as a Signal sequences. To achieve high-level expression, we cloned the in Glucose-Stimulated Insulin Secretion mitochondrially targeted S100G into an adenovirus vector under The main physiological regulator of insulin secretion is glucose, the control of the doxycycline (DOX)-inducible tetON promoter to which promotes oxidative metabolism, mitochondrial respira- generate Ad-mitoS100G. After infection with Ad-mitoS100G and tion, mitochondrial ATP synthesis, and the generation of addi- Ad-tetON, cells displayed DOX-dependent mitoS100G expres- tional coupling factors (Wiederkehr and Wollheim, 2008). In sion (Figure 1A) reaching a maximum within 24 hr (1 mg/ml turn, the cytosolic ATP:ADP ratio is elevated, closing KATP chan- DOX; Figure 1B). Neither adenovirus infection nor high-level nels and eliciting plasma membrane electrical activity. Cytosolic expression of mitoS100G caused any apparent changes in cell Ca2+ transients caused by voltage-gated Ca2+ influx are relayed survival (Figure S1, available online). Proper mitochondrial into mitochondria (Kennedy et al., 1996). During glucose stimula- 2+ targeting of S100G is suggested from the two protein bands. tion of INS-1E cells, [Ca ]m rose from 222 ± 14 nM to 875 ± The upper faint band of about 15 kDa is full-length mitoS100G. 30 nM in control infected cells (Figure 3A). Expression of 2+ The prominent band at 9 kDa is the S100G protein after import mitoS100G did not influence basal [Ca ]m (213 ± 17 nM). and removal of the targeting sequence by mitochondrial pro- MitoS100G markedly reduced the amplitude of the response cessing peptidase. Targeting of mitoS100G was confirmed by (531 ± 48 nM; n = 3; p = 0.0039; Figure 3A). The onset of the 2+ confocal microscopy in INS-1E cells (rat insulinoma cells). glucose-stimulated [Ca ]m increase in INS-1E cells is relatively Galleries of Z stacks taken from INS-1E cells expressing rapid (19.3 ± 4.1 s) and not significantly delayed after expression 2+ mitoS100G and 3D reconstruction demonstrate localization to of mitoS100G (21.7 ± 5.2 s). The average [Ca ]m rise often a filamentous network typical for mitochondrial (Fig- appeared biphasic (Figure 3A), a phenotype also visible in indi- ure 1C and Figure S2). Confocal images of S100G-positive vidual experiments (see Figure S3). After expression of the filamentous structures closely colocalized with mitotracker in control proteins mitoS100A1 or mitoECFP the amplitude of the H295R cells (human adrenocortical cancer, Figure 1D). Mito- mitochondrial Ca2+ response was unchanged (Figure 3C and chondrial targeting was also observed in HeLa cells (human Figure S3). Glucose-stimulated insulin secretion was not epithelial cancer line, data not shown). affected by mitoS100A1 (Figure 3D). On the other hand, 2+ Two control proteins were targeted to the mitochondria, the mitoS100G, in addition to its ability to buffer [Ca ]m, caused 2+ low-affinity Ca -binding protein S100A1 (KD 290 mM; Maren- a pronounced reduction of glucose-stimulated insulin secretion holz et al., 2004) and mitoECFP (Figure S2). An earlier study (Figure 3B). This inhibition was dependent on the concentration had found S100A1 to localize to mitochondria where it controlled of DOX used to induce expression of mitoS100G (Figure S3). 2+ ATP synthesis in cardiomyocytes (Boerries et al., 2007). In The results show that [Ca ]m is a signal necessary for efficient INS-1E cells, S100A1 did not localize to mitochondria. Further- metabolism-secretion coupling.

602 Cell Metabolism 13, 601–611, May 4, 2011 ª2011 Elsevier Inc. Cell Metabolism Signaling Function of Matrix Ca2+

ABno virus MitoS100G Figure 1. Expression and Targeting of S100G to the Mitochondrial Matrix no virus MitoS100G doxycycline doxycyline Insulinoma INS-1E (A–C) or adrenocortical H295R (ng/ml) 0 0 8 1000 40 200 1000 5000 24 840 2416 48 induction (hours) cells (D) were infected with adenoviruses for the 15 15 expression of mitoS100G. MitoS100G MitoS100G (A and B) Protein extracts were analyzed by 6 6 western blotting. mitoS100G was induced with increasing concentrations of DOX for 24 hr (A) or 37 37 GAPDH GAPDH for different times in the presence of 1 mg/ml DOX (B). The blots were probed with an anti-S100G (upper panels) and reprobed with an anti-GAPDH C antibody (lower panels). (C and D) Cells were stained with anti-S100G (red). H295R cells were loaded with MitoTracker dye for 30 min prior to fixation (D; green). Colocalization is shown in yellow. Scale bar = 5 mm. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. See also Figures S1 and S2.

2+ vidual cells) the amplitude of the [Ca ]c increase is rather small and was not affected when matrix Ca2+ was buffered after expression of mitoS100G (Fig- ure 4A). To improve the sensitivity, we also performed kinetic analysis of Ca2+ D transients at the single cell level by using the Ca2+ sensor YC3.6 cloned into an adenovirus vector (Ad-RIP-YC3.6; Figures 4B–4E). Previously, this sensor and similar protein-based Ca2+ probes have been successfully applied for the study of cytosolic Ca2+ signals in INS-1E and primary mouse b cells (Akhmedov et al., 2010; Ravier et al., 2010). Expres- sion of mitoS100G in YC3.6-positive cells was confirmed by immunofluorescence (Figure 4E). Glucose stimulation resulted in cytosolic Ca2+ transients in 71% (22/ 31) of control cells and in 72% (31/43) of the mitoS100G-expressing cells (from six independent experiments). The frequency of individual Ca2+ spikes after glucose was not significantly changed In contrast to glucose and other fuel secretagogues, K+- by mitochondrial Ca2+ buffering (Figures 4B–4D). Furthermore, 2+ 2+ induced [Ca ]c rises stimulate insulin secretion independently the time of onset of the [Ca ]c rise did not differ between the of mitochondrial function as also demonstrated in insulinoma two experimental groups (Figure 4A and data not shown). rho0 cells (Kennedy et al., 1998). Consistent with these findings, Thus, inhibition of the glucose-dependent mitochondrial Ca2+ K+-induced insulin secretion was unaffected when mitochondrial rise is not the secondary consequence of impaired cytosolic Ca2+ signals were buffered by using mitoS100G (Figure 2F). Ca2+ signals. Furthermore, matrix Ca2+ buffering lowers insulin secretion by a mechanism that is not directly linked to the modu- Matrix Ca2+ Buffering Does Not Reduce lation of cytosolic Ca2+. 2+ Glucose-Induced [Ca ]c Rises in INS-1E Cells Cytosolic Ca2+ signals act as the principal trigger for insulin Matrix Ca2+ Signals Accelerate Glucose-Dependent 2+ granule exocytosis. Therefore under most conditions, [Ca ]c Respiration and ATP Synthesis rises and insulin exocytosis are closely correlated. To investigate The importance of Ca2+ as an activating signal in the mitochon- 2+ 2+ this, we measured [Ca ]c with aequorin. Monitored under the drial matrix has been proposed based on the observed Ca same conditions, the kinetics of cytosolic and mitochondrial dependence of oxidative metabolism and mitochondrial ATP Ca2+ responses are quite similar (Kennedy et al., 1996; Figures synthesis (Hajno´ czky et al., 1995; Hellman et al., 1974; Kennedy 3A and 4A). In these population experiments (average of indi- et al., 1999; McCormack et al., 1990; Pralong et al., 1992, 1994).

Cell Metabolism 13, 601–611, May 4, 2011 ª2011 Elsevier Inc. 603 Cell Metabolism Signaling Function of Matrix Ca2+

ABCFigure 2. Specific Buffering of Mitochon- histamine histamine histamine drial Matrix Ca2+ Rises with mitoS100G 3000 1200 4000 control control control (A–C) HeLa cells were perifused in KRBH buffer mitoS100G mitoS100G mitoECFP containing 5.5 mM glucose and stimulated with 1 mM histamine. 3000 (D–F) INS-1E cells were perifused in KRBH buffer 2+ 2000 2+ 800 2+ 2+ containing 2.5mM glucose. Ca influx was stim- ulated by using 30 mM KCl. 2000 (A–F) Cells were infected with adenoviruses for the expression of mitoS100G (A, B, and D–F) or mi- 1000 400 toECFP (C) and either an adenovirus carrying Cytosolic Ca (nM) 1000

Mitochondrial Ca (nM) Mitochondrial Ca (nM) mitochondrial aequorin (A, C, and D) or cytosolic aequorin (B and E). After infection, the cells were cultured for 24 hr in the presence of 1 mg/ml DOX 0 0 0 0 200 400 0 200 400 0 200 400 (induction of mitoS100G or mitoECFP; gray traces) time (seconds) time (seconds) time (seconds) or absence of DOX (control; black traces). For experiments with HeLa cells the aequorin variants DF30mM KCl E 30mM KCl 1200 were expressed constitutively driven by the control control p=0.42 chicken actin promoter. The average ± SEM from mitoS100G 3000 mitoS100G 4 n = 5 (A), n = 4 (B, C, and E), and n = 3 (D and F) experiments is shown. See also Figure S2. (F) Static insulin secretion. Prior to the experi- 2+ 800

2000 2+ ments, INS-1E cells were infected with Ad-mi- 2 toS100G in combination with Ad-tetON and

% of content cultured for 24 hr in the presence or absence of 400 insulin secretion 1 mg/ml DOX. The cells were incubated for 30 min 1000 

Cytosolic Ca (nM) at 37 C in KRBH containing 2.5 mM glucose or

Mitochondrial Ca (nM) 0 stimulated with 30 mM KCl. 30mM KCl -+-+ 0 0 doxycycline - -+ + 0 200 400 0 200 400 time (seconds) time (seconds)

Consistent with this type of regulation, we observed pronounced respiratory rate of INS-1E cells doubled rapidly (0–9 min) during inhibition of oxidative phosphorylation when Ca2+ signals were glucose stimulation followed by a slower second phase of attenuated (Figures 5A and 5D). Under control conditions, the continuous increase of the respiratory rate over the next

2+ Figure 3. Buffering [Ca ]m Rises Lowers AB2.5mM 16.7mM glucose 1000 mitoS100G Glucose-Stimulated Insulin Secretion control (A–D) INS-1E cells were infected with Ad-mi- MitoS100G p=0.001 800 8 toS100G in combination with Ad-tetON (A and B) or Ad-mitoS100A1 plus Ad-tetON (C and D) in

2+ 6 2+ 600 combination with Ad-mitoAequorin for [Ca ]m measurements (A and C). After infection, the cells 4 400 were grown in INS medium in the absence (control

% of content cells, black traces) or presence of 1 mg/ml DOX insulin secretion 2 (induced transgene, gray traces) for 24 hr. Cells 200

Mitochondrial Ca (nM) were stimulated by raising glucose from 2.5 to 0 2.5 16.7 16.72.5 mM glucose 16.7 mM. For clarity, error bars are only shown 0 0 300 600 900 1200 --++doxycycline every 120 s. (A–D) Average ± SEM; n = 3. time (seconds) (B and D) Static insulin secretion experiments. CD2.5mM 16.7mM glucose Insulin secretion and content were measured in 1200 mitoS100A1 control 8 INS-1E cells expressing mitoS100G (B) or MitoS100A1 p=0.609  1000 mitoS100A1 (D) after incubation for 30 min at 37 C 6 in KRBH containing either 2.5 mM or 16.7 mM 800 glucose. See also Figure S3. 2+ 600 4 % of content

400 insulin secretion 2

200 0 Mitochondrial Ca (nM) 2.5 16.7 16.72.5 mM glucose 0 --++doxycycline 0 300 600 900 1200 time (seconds)

604 Cell Metabolism 13, 601–611, May 4, 2011 ª2011 Elsevier Inc. Cell Metabolism Signaling Function of Matrix Ca2+

2+ A 2.5mM 16.7mM glucose Figure 4. Mitochondrial Matrix Ca Buff- 120 ering Does Not Inhibit Cytosolic Ca2+ control Responses after Glucose Stimulation mitoS100GMitoS100G 90 (A–E) INS-1E cells were infected with Ad-mi- toS100G in combination with Ad-tetON and Ad- 60 RIP-cytoAequorin (A) or Ad-RIP-YC3.6 (B–E) to 2+ measure [Ca ]c. After infection, INS-1E cells were

2+ cultured for 24 hr with (mitoS100G; gray trace) or 30 without (control; black trace) 1 mg/ml DOX. The 2+ average glucose-induced increase of [Ca ]c from 0 three independent experiments (± SEM) is shown. 2+ Cytosolic Ca Increase (nM) (B and C) Ca transients in response to glucose in a control (B) or mitoS100G expressing cell (C). 06001200 time (seconds) (D) The average peak frequency at 2.5 mM, 16.7 mM glucose, and in response to 200 mM B 2.5mM 16.7mM glucose 2.5mM C 2.5mM 16.7mM glucose 2.5mM tolbutamide (average ± SEM; n = 6). A total of 31 control infected cells and 43 mitoS100G-ex- 1.6 1.6 pressing cells were analyzed. (E) MitoS100G (left, red) expression in YC3.6- 1.4 1.4 positive cells (right, yellow) was confirmed by immunocytochemistry. Scale bar = 5 mm. 1.2 1.2 ratio 535/480 ratio 535/480 1 1 n = 4; p = 0.05). Furthermore, when mi- toS100G was expressed, hyperpolariza- 0 600 1200 0 600 1200 time (seconds) time (seconds) tion of the inner mitochondrial membrane D p=0.33 E in response to glucose stimulation was 1.2 similar to the control during the first p=0.29 ** 6 min but was reduced at later time points * 2+ * p=0.008 (Figure 5F). Therefore, the matrix Ca 0.8 signal becomes important only after the ** p=0.006 initial acceleration of respiration, net 0.4 ATP increases, and hyperpolarization of

peaks/ minute cell the inner mitochondrial membrane.

5μm 0 Matrix Ca2+ Is a Potentiating Signal 2.516.72.5 2.5 16.7 2.5 mM glucose μ 200 200 M tolbutamide mitoS100G YC3.6 for Second-Phase Insulin Secretion 111000 μg/ml doxycycline in Primary b Cells The matrix Ca2+ signal affects energy metabolism mostly beyond the time window of the matrix Ca2+ rise (compare 40 min (Figures 5A and 5B). This time course was unaffected by Figures 3 and 5), suggesting a main role in sustained glucose- virus infection (transgene not induced; Figure 5B). After removal induced insulin secretion. These results are also consistent of extracellular Ca2+, which abolishes glucose-dependent Ca2+ with earlier findings that demonstrate a priming role for Ca2+ in signals, the initial acceleration of respiration was close to normal the activation of mitochondrial ATP production (Jouaville et al., but the subsequent further increase of the respiratory rate was 1999). To test whether such a feed-forward mechanism is indeed blunted (Figure 5A). Induction of mitoS100G caused a marked operative, we studied primary b cells, which have a more clearly reduction of glucose-induced respiration primarily affecting the defined biphasic insulin secretion than INS-1E cells (Akhmedov second phase of the response (Figure 5C). This result demon- et al., 2010; Wiederkehr et al., 2009)(Figure 6C). Furthermore, strates that matrix Ca2+ is an activating signal for mitochondrial by using this system, effective adenovirus infection of islet b cells respiration. is easier to achieve than with intact islets. In control infected 2+ As a consequence of the reduced respiratory response the net cells, glucose stimulation resulted in a [Ca ]m rise reaching ATP increase observed in the cytosol during glucose stimulation a maximal value of 781 ± 38 nM (n = 5; Figure 6A). After expres- was blunted when Ca2+ influx was prevented (Figure 5D) or sion of mitoS100G the amplitude was strongly reduced to 271 ± 2+ [Ca ]m was buffered with mitoS100G (Figure 5E). The kinetics 56 nM (n = 5; p = 0.0006; Figure 6A). During the second phase 2+ 2+ of the ATP response was monitored by following the lumines- [Ca ]m was only slightly elevated compared to Ca ]m under cence of cytosolic luciferase. Matrix Ca2+ buffering by using mi- basal conditions. However, this plateau was significantly toS100G lowered the rate of ATP synthesis during the second reduced when mitoS100G was expressed (p = 0.045; Figure 6B). 2+ phase of the response from1.05 ± 0.25%/min (control cells Similarly, the [Ca ]m rises after plasma membrane depolariza- infected but not induced with DOX) to 0.4 ± 0.15%/min in cells tion with 30 mM KCl were blunted when mitoS100G was ex- expressing mitoS100G (basal luminescence set to 100%; pressed (Figures 6A and 6B).

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ABC2.5mM 16.7mM glucose 2.5mM 16.7mM glucose 2.5mM 16.7mM glucose 2000 600 uninduced control doxycycline 700 1600 500 600 400 1200 500 400 300 800 300 200 pmoles/ minute pmoles/ minute pmoles/ minute 200 400 control no virus no virus 100

oxygen consumption rate 100 oxygen consumption rate oxygen consumption rate EGTA mitoS100G mitoS100G 0 0 20 40 60 0 20 40 60 80 0 20 40 60 80 time (minutes) time (minutes) time (minutes)

D 2.5mM 16.7mM glucose EF2.5 16.7mM glucose 2.5mM 16.7mM glucose 130 130 160 FCCP

120 120 120 FCCP - R 110 110 80 uninduced control basal mitoS100G cytosolic ATP cytosolic ATP cytosolic ATP cytosolic ATP 40 100 100 fluoresence ratio

control uninduced control % of R EGTA % of signal at basal glucose

% of signal at basal glucose mitoS100G 0 90 90 0 600 1200 1800 2400 0 600 1200 0 600 1200 time (seconds) time (seconds) time (seconds)

Figure 5. Mitochondrial Matrix Ca2+ Signals Are Required for the Sustained Activation of Mitochondrial Energy Metabolism during Glucose Stimulation of INS-1E Cells Glucose-mediated Ca2+ signaling in INS-1E cells was either suppressed by removing extracellular Ca2+ (KRBH without added Ca2+ plus 0.4 mM EGTA; A and D) or specifically buffered in the mitochondria by using mitoS100G (C, E, and F). Respiration rates (A–C) were measured every 9 min. (A) Cells were stimulated with 16.7 mM glucose in the presence (filled diamonds) or absence of extracellular Ca2+ (empty squares). Because of variations in the absolute respiration rates, one typical experiment of three independent experiments performed in quadruplicate is shown (average ± SEM). (B) Glucose-induced respiratory response of control uninfected INS-1E cells (filled diamonds) or 24 hr after infection with Ad-mitoS100G in combination with Ad- tetON (empty squares) in the absence of DOX. (C) As described for (B) but in the presence of DOX (1 mg/ml) for 24 hr. (B and C) The average of three independent experiments (± SEM), each performed in quadruplicate, is shown. (D) Glucose-induced ATP changes in control (black trace) and after chelation of extracellular Ca2+ (gray trace). (D and E) For cytosolic ATP measurements, INS-1E cells were coinfected with Ad-RIP-Luciferase. Representative examples of four independent experiments are shown. (E) Glucose response 24 hr after infection with Ad-mitoS100G in combination with Ad-tetON cultured with (gray trace) or without (black trace) 1 mg/ml of DOX. (F) Glucose-dependent hyperpolarization of the mitochondrial membrane potential measured with JC-1. Virus infection without induction of the transgene (control; upper black trace) or after 24 hr of mitoS100G expression (gray trace). Lower black trace: infected cells maintained at 2.5 mM glucose. FCCP (10 mM) was added to depolarize the mitochondrial membrane potential (mean ± SEM; n = 3).

The impact of matrix Ca2+ buffering on insulin secretion was Mitochondrial Ca2+ Regulates Angiotensin II-Induced studied in samples taken from the effluent of cell preparations NAD(P)H Formation and Aldosterone Secretion 2+ during [Ca ]m measurements. In control infected cells insulin Aldosterone secretion from glomerulosa cells of the adrenal secretion reached a first maximum 3.5 min after initiation of cortex is another physiological process tightly linked to Ca2+ the glucose response (first phase) and returned to intermediate signaling. The most important physiological stimuli of aldoste- levels of insulin secretion before rising again (second phase). rone secretion are K+ and angiotensin II (Spa¨ t and Hunyady, The kinetics of hormone secretion were similar in rat islet 2004). Whereas K+ activates voltage-dependent Ca2+ channels, b cells expressing mitoS100G but secretion was clearly reduced angiotensin II mobilizes Ca2+ from the ER with a subsequent during the second phase (À49%; n = 5; p = 0.034; Figures influx of Ca2+ from the extracellular space. Cytosolic and mito- 6C and 6D). First-phase insulin secretion and the secretory chondrial Ca2+ signals were analyzed in human aldosterone- response of b cells to 30 mM KCl were not significantly inhibited producing (glomerulosa-like) H295R cells after loading with the (Figure 6D). We conclude that the matrix Ca2+ signal is required Ca2+ indicators Fluo-4 (cytosolic Ca2+) and Rhod-2 (mitochon- for the potentiation of glucose-induced second-phase insulin drial Ca2+)(Szanda et al., 2008). Angiotensin II evoked 2+ 2+ secretion. a [Ca ]c rise and an accompanying [Ca ]m response (Figure 7A).

606 Cell Metabolism 13, 601–611, May 4, 2011 ª2011 Elsevier Inc. Cell Metabolism Signaling Function of Matrix Ca2+

2+ A B Figure 6. Matrix Ca Buffering in Primary 2.5mM 16.7mM glucose KCl Rat Islet Cells Blunts Second-Phase Insulin 1200 1000 p<0.001 p=0.003 Secretion

2+ Substrate-attached primary rat islet cells were 800 infected with Ad-mitoS100G in combination with

[nM] 800 Ad-tetON and Ad-mitoAequorin 24 hr before the 2+ 2+ 600 experiment. Islet cells were perifused and [Ca ]m p=0.045 (A and B) and insulin secretion (C and D) were 400 measured on the same cell preparations. Islet cells 400 were stimulated with 16.7 mM glucose. Depolar- area under curve mitochondrial Ca ization was induced with 30 mM KCl. 0 123 200 2+ dox -+ -+ -+ (A) [Ca ]m responses were measured in control

mitochondrial Ca islet cells (without induction of mitoS100G; black 0 trace) or expressing mitoS100G (1 mg/ml DOX; 0 600 1200 1800 2400 24 hr; gray trace). For clarity standard error bars time (seconds) 1st phase 2nd phase30mM KCl are only shown every 120 s. 2+ 2.5mM 16.7mM glucose KCl (B) Quantification of the [Ca ]m response (area C D under the curve). 5 (C) Insulin concentration in fractions during islet 100 p=0.034 cell perifusion described in (A). 4 (D) Quantification of insulin secretion. First phase: 80 insulin secreted during 6 min after initiation of the 3 60 glucose response. Second phase: sum 12–24 min p=0.28 after initiation of the glucose response. 30mM

ng/ml p=0.26 2 40 KCl: the sum of insulin secreted during 4 min of KCl-induced depolarization is shown.

insulin secretion 1 20 (B and D) Black bars show control and gray bars m

insulin secretion [ng] show values after induction of mitoS100G (1 g/ml 0 0 of DOX). The average ± SEM of five independent 0600 1200 1800 2400 dox -+ -+ -+ experiments is shown. time (seconds)

1st phase 2nd phase30mM KCl

Expression of mitoS100G revealed highly significant reduction of a basal value of 397 ± 54 to 1299 ± 128 pg/mg protein/2 hr. In the angiotensin II-induced mitochondrial Ca2+ signal (p = cells expressing mitoS100G, hormone secretion was reduced 2+ 0.0014), while the [Ca ]c response was moderately increased (basal value 293 ± 29, stimulated value 977 ±122 pg/mg (p = 0.0026; Figure 7B). Expression of mitoECFP in the mitochon- protein/2 hr; for the effect of mitoS100G: p = 0.020; n = 6 in all drial matrix of H295R cells had no effect on cytosolic or mito- 4 groups, Figure 7D). Expression of mitoECFP did not affect chondrial Ca2+ signals in response to angiotensin II (data not the hormonal response to angiotensin II (data not shown). The shown). present data demonstrate the importance of intramitochondrial Aldosterone secretion reflects the rate of hormone synthesis Ca2+ both in basal and stimulated aldosterone production. from cholesterol, which is regulated by cAMP and Ca2+. Both messengers activate the transport of cholesterol across the inner DISCUSSION mitochondrial membrane (Cherradi and Capponi, 1998; Spa¨ t and Hunyady, 2004). Here, matrix enzymes catalyze side-chain The main role attributed to the mitochondrial matrix Ca2+ rise is cleavage and most of the following reactions resulting in aldoste- the acceleration of oxidative metabolism and ATP production rone synthesis. This biosynthetic pathway depends on the to balance the increased ATP demand caused by Ca2+-depen- generation of mitochondrial NADH to form NADPH. NADPH dent energy-consuming processes. Ca2+ is thought to influence works together with molecular oxygen as a cofactor in the mitochondrial metabolism either by stimulating Ca2+-sensitive hydroxylation of intermediates in aldosterone biosynthesis. The matrix dehydrogenases (McCormack et al., 1990) or by binding Ca2+-mobilizing hormone angiotensin II enhances the reduction to EF hand-containing proteins in the intermembrane space of NAD+ and NADP+ (Pralong et al., 1994; Roha´ cs et al., 1997). (Ma´ rmol et al., 2009; Perocchi et al., 2010). Consistent with these earlier findings, the NAD(P)H level rose shortly after angiotensin II stimulation (Figure 7C). Compared mitoS100G Buffers Mitochondrial Matrix Ca2+ Signals to control cells, expression of mitoS100G lowered the Ca2+- Here, we have expressed the Ca2+-binding protein S100G in the dependent NAD(P)H response to angiotensin II (p = 0.0092; Fig- mitochondria to specifically buffer the matrix Ca2+ rise either ure 7C). Because of a possible cytosolic contamination of the after mobilization of Ca2+ from the ER or after inducing Ca2+ influx mitochondrial NAD(P)H signal the effect of the matrix Ca2+ buff- from the extracellular space in different cell types. In contrast, ering on the NAD(P)H response may be underestimated. inhibitors such as ruthenium red or Ru360 block mitochondrial 2+ 2+ Similarly, expression of mitoS100G affected angiotensin II- Ca uptake and therefore also directly alter [Ca ]c responses. dependent hormone secretion. In infected cells not exposed to In addition ruthenium red has side effects on other Ca2+ trans- DOX, angiotensin II augmented aldosterone secretion from porters in the ER and the plasma membrane (Hajno´ czky et al.,

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2+ ABin reduced negative feedback exerted by high [Ca ]onIP3 6 p=0.0026 2+ receptors (Bezprozvanny et al., 1991). Ca mito 6 p=0.0014

NAD(P)H Responses and Aldosterone Secretion Depend 0

0 4 on [Ca2+] Signals 4 m Expression of mitoS100G decreased the NAD(P)H response to

2+ 2+ 2+ Ca cyto the Ca raising hormone angiotensin II. Aldosterone secretion Ca F/F 2 calcium F/F 2 was reduced in angiotensin II-stimulated as well as nonstimu- lated glomerulosa cells. The latter reduction is compatible with the observation that constitutive IP3R-mediated Ca2+ transfer 0 0 0 60 120 180 to mitochondria may be essential for efficient mitochondrial metabolism even under resting conditions (Ca´ rdenas et al., time (seconds) control 2010). This reduction clearly illustrates the requirement of matrix mitoS100G Ca2+ for the stimulation of mitochondrial dehydrogenases. Of CD note, aldosterone secretion was diminished despite the [Ca2+] 1600 c p<0.0001 elevation. 0.3 NADPH formation is linked to NADH by mitochondrial nucleo- 1200 tide transhydrogenases and acts as a cofactor in reductive biosynthesis such as steroid hydroxylation. TCA intermediates 0.2 800 support not only the reduction of mitochondrial pyridine nucleo- tides but also steroid hydroxylation in glomerulosa cells Δ NAD(P)H 0.1 (reviewed in Spa¨ t and Hunyady, 2004). Moreover, NADPH is 400 not in excess but is utilized during increased synthesis of aldo- sterone (Roha´ cs et al., 1997). Nevertheless, the significance of 2+ 0 Aldosterone/ mg protein/ 2h pg 0 the matrix Ca -stimulated NADPH formation in the control of hormone secretion has been neglected, focusing instead on control control Ca2+ activation of StAR, which mediates cholesterol uptake mitoS100G mitoS100G (Cherradi and Capponi, 1998). Here we provide unequivocal evidence for the importance of matrix Ca2+ signals in the stimu- 2+ Figure 7. mitoS100G Lowers the Angiotensin II-Stimulated [Ca ]m lation of reductive biosynthesis via the regulation of matrix dehy- Response, NAD(P)H Formation, and Aldosterone Secretion drogenases, as exemplified by aldosterone synthesis. H295R cells were infected with Ad-mitoS100G in combination with Ad-tetON and loaded 48 hr later with Fluo-4 (cytosolic Ca2+) and Rhod-2 (mitochondrial [Ca2+] Signals Are Required for Sustained Activation Ca2+). m (A) Cytosolic (black trace) and mitochondrial (gray trace) Ca2+ rises in response of Glucose-Dependent Respiration and ATP Synthesis to angiotensin II (1 nM) were measured simultaneously in individual cells. In the present study, we have obtained insights into the kinetics Maximal fluorescence changes were expressed as fold increase over basal of matrix Ca2+-dependent acceleration of oxidative phosphory- values for both parameters. lation in insulin-secreting cells. Initial increases of the respiratory (B) Analysis of average cytosolic (black bars) and mitochondrial (gray bars) rate and net ATP changes in the cytosol were little affected by Ca2+ responses to angiotensin II. Control infected cells (n = 10) were compared [Ca2+] buffering. During the subsequent sustained acceleration to cells expressing mitoS100G (n = 20) after 48 hr induction with 1 mg/ml of m DOX. of respiration and net ATP increases, mitoS100G expression (C) NAD(P)H autofluorescence increases in response to 1 nM angiotensin II blunted the glucose response. The initial activation of energy were measured in control (33 cells) and mitoS100G-expressing cells (33 cells). metabolism is explained by enhanced provision of glycolysis- (D) Basal (black bars) and angiotensin II-stimulated hormone secretion derived pyruvate (reviewed in Wiederkehr and Wollheim, 2008). (gray bars) was measured from control infected cells and cells after induction The associated early rise of ATP is required to induce closure (1 mg/ml DOX) of mitoS100G for 48 hr (n = 6). of K channels for the initiation of action potentials and cyto- (A–D) The average ± SEM is shown. ATP solic Ca2+ transients (triggering pathway; Henquin, 2009). We show that the subsequent acceleration of respiration and ATP 2006). Furthermore, protonophores depolarizing the mitochon- generation depends on matrix Ca2+ signals. The division of the drial membrane potential, while inhibiting mitochondrial Ca2+ mitochondrial activation into a Ca2+-independent and Ca2+- 2+ uptake, also augment [Ca ]c (Babcock et al., 1997). dependent phase is also consistent with studies describing 2+ 2+ To test for effects unrelated to [Ca ]m buffering, we expressed NAD(P)H responses prior to the onset of cytosolic Ca signals mitoECFP and mitoS100A1, confirming the specificity of the mi- (Luciani et al., 2006; Pralong et al., 1990). Ca2+ can only poten- toS100G approach. MitoS100G reduced the matrix Ca2+ rises tiate mitochondrial activity in the b cell once metabolism has 2+ 2+ without lowering the [Ca ]c in INS-1E and HeLa cells. In caused a first round of cellular activation leading to Ca influx. 2+ H295R cells we observed a moderate increase in [Ca ]c after 2+ angiotensin II stimulation. The increased [Ca ]c response may Amplification of Glucose-Stimulated Insulin Secretion be attributed to the enhanced mitochondrial buffering of Ca2+, Is Regulated by Mitochondrial Matrix Ca2+ Signals leading to a diminished Ca2+ signal in the microdomain between MitoS100G attenuated glucose-stimulated insulin release the mitochondria and apposing ER membranes, finally resulting despite normal cytosolic Ca2+ transients. Lowered insulin

608 Cell Metabolism 13, 601–611, May 4, 2011 ª2011 Elsevier Inc. Cell Metabolism Signaling Function of Matrix Ca2+

secretion in the face of normal cytosolic Ca2+ signals suggests sodium pyruvate, 50 mg/ml penicillin, and 100 mg/ml streptomycin (INS that the amplifying pathway of insulin secretion is inhibited. medium). H295R cells (NCI-H295R; CRL-2128, lot number 58209026; ATCC, This latter process is believed to depend on mitochondrially Manassas, VA) were grown in DMEM/Ham’s F12 (1:1, vol:vol) containing 1% ITS+, 2% UltroSer G, 100 U/ml penicillin, and 100 mg/ml streptomycin. derived metabolites including glutamate, citrate, and NAD(P)H Passage numbers 4–10 were used. HeLa cells were cultured in DMEM medium (Ivarsson et al., 2005; Joseph et al., 2006; Maechler and Woll- containing 5.5 mM glucose, 4 mM L-glutamine, and 1 mM sodium pyruvate. heim, 1999). Here we demonstrate that the molecular events Dispersed rat islet cells were obtained after digestion of islets in trypsin linking matrix Ca2+ signals to the amplification of insulin secre- EGTA solution (GIBCO; Switzerland) (Kirkpatrick et al., 2010). tion are operative in primary b cells. The pronounced reduction 2+ of the [Ca ]m rise in rat b cells reduced second-phase insulin Recombinant Adenoviruses secretion preferentially. This may be surprising as the control The adenoviruses expressing cytoplasmic luciferase (Ad-RIP-Luciferase), 2+ cytosolic aequorin (Ad-RIP-Aequorin), and mitochondrially targeted aequorin [Ca ]m rise is essentially monophasic followed by a small 2+ (Ad-RIP-mitoAequorin) were constructed as described (Ishihara et al., 2003). plateau. The matrix Ca signal is thus probably an initiating 2+ The Ca sensor YC3.6 was cloned behind the rat insulin promoter into an event preparing mitochondria for sustained activation by nutri- adenovirus vector (Clontech, CA) (Akhmedov et al., 2010). S100G and ents. Similarly, in HeLa cells, prestimulation with histamine S100A1 cDNAs were fused to a mitochondrial targeting sequence and cloned causing cytosolic and mitochondrial Ca2+ rises enhances ATP into an adenovirus vector under the control of the tetON promoter (Ad-mi- production in response to subsequent metabolic activation, toS100G and Ad-mitoS100A1). a phenomenon the authors termed long-term metabolic priming (Jouaville et al., 1999). Alternatively, in b cells Ca2+ cycling Luminescence Measurements 2+ 2+ responsible for the plateau phase may contribute to stimulation [Ca ]c, [Ca ]m, and cytosolic ATP measurements were performed as b 2+ described (Ishihara et al., 2003; Kennedy et al., 1996; Wiederkehr et al., of metabolism during sustained activation. In the cell, [Ca ]m 2009). One day after plating INS-1E or dispersed rat islet cells were infected rises are thought to enhance the release of coupling factors for 90 min at 37C with Ad-mitoS100G or Ad-mitoS100A1 in combination from mitochondria, which are required to potentiate granule with the tetON transactivator virus (Ad-tetON) and the reporter virus Ad-Lucif- m exocytosis independent of the regulation of the KATP channel erase, Ad-Aequorin, or Ad-mitoAequorin. DOX (1 g/ml) was added to the or the modulation of cytosolic Ca2+ signals (Ivarsson et al., medium to induce the transgene. All measurements on INS-1E or dispersed 2005; Joseph et al., 2006; Maechler and Wollheim, 1999). In rat islet cells were performed in Krebs-Ringer bicarbonate HEPES buffer (KRBH) containing 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH PO , 0.5 mM fact, recent evidence shows that amplification does not occur 2 4 2+ MgSO4, 1.5 mM CaCl2, 10 mM HEPES, 5 mM NaHCO3, and 2.5 mM glucose via enhancing subplasma membrane Ca signals (Ravier (pH 7.4). The cells were perifused at a rate of 1 ml/min. For kinetic insulin secre- et al., 2010). First-phase insulin secretion appears to be mini- tion experiments fractions were taken every 30 s. mally dependent on the factors amplifying the effect of cytosolic 2+ Ca on the second phase. Of note, blunting of matrix alkaliniza- Cytosolic Ca2+ Imaging tion inhibits second-phase insulin secretion as well (Akhmedov Cytosolic Ca2+ transients were measured by using Ad-RIP-YC3.6 as et al., 2010). Amplification of insulin secretion may thus depend described (Akhmedov et al., 2010). Image acquisition was performed on an on the dual control of mitochondrial function by matrix Ca2+ and inverted microscope (Zeiss Axiovert 200 M, Carl Zeiss AG, Switzerland) with an array laser confocal spinning disk (QLC100; VisiTech, UK). Cells were matrix alkalinization. imaged by using a 63 3 (numerical aperture 1.4) oil-immersion objective (Carl Zeiss AG). YC3.6 was excited with 440 nm light and emission was Conclusions followed at 480 and 535 nm. Images were acquired every 2 s and analyzed Mitochondria are able to decode the frequency and amplitude of by using Metafluor 6.3 software (Universal Imaging, Molecular Devices cytosolic Ca2+ transients, a mode of communication that is still Corporation; USA). poorly understood. Here we demonstrate that reducing the Simultaneous Measurement of [Ca2+] and [Ca2+] amplitude of matrix Ca2+ rises slows oxidative metabolism, c m H295R cells (2 3 104) were plated onto 24 mm diameter circular glass cover- respiration, and ATP synthesis with effects on metabolism- slips and infected 1 day later (60 IFU/cell of mitoS100G and 30 IFU/cell of te- secretion coupling in insulin-secreting cells and steroidogenesis tON). Immunocytochemical examination revealed the expression of S100G in 2+ in glomerulosa cells. Taken together, our results reveal a physio- 63% of the cells. Two days later simultaneous measurement of [Ca ]c with 2+ 2+ logical importance of [Ca ]m rises in these processes and Fluo-4 and [Ca ]m with Rhod-2 was performed in H295R superfusion medium: 140 mM NaCl, 3.1 mM KCl, 1.2 mM CaCl2, 0.5 mM KH2PO4, 0.5 mM MgSO4, emphasize their pivotal role as intermediate signals modulating  cellular responses to hormones and nutrients. 5 mM Na-HEPES, 2 mM NaHCO3, and 12 mM glucose (pH 7.4) at 35 C (flow rate 1 ml/min) with a confocal laser scanning microscopy as described (Szanda et al., 2008). EXPERIMENTAL PROCEDURES 2+ Simultaneous NAD(P)H and [Ca ]c Measurements Most chemicals and reagents were from Sigma and Fluka Chemie 2+ Simultaneous measurement of [Ca ]c and mitochondrial NAD(P)H was carried (Switzerland). Coelenterazine was purchased from Calbiochem. Beetle lucif- out with an inverted microscope (Axio Observer, Zeiss) equipped with a erin was obtained from Promega (Switzerland). Fluo-4, Rhod-2, and JC-1 40 3 oil immersion objective (Fluar, Zeiss) and a Cascade II camera (Photo- were from Invitrogen (Paisley, UK). Polyclonal antibodies against S100G metrics) was used. Excitation wavelengths were set by a random access (CB9; Swant, Switzerland), S100A1 (SP5355P; Acris, Germany) and GAPDH monochromator connected to a xenon arc lamp (DeltaRAM, Photon Tech- (G9545; Sigma) were used. 2+ nology International). For monitoring [Ca ]c Fluo-4 was excited at 488 nm and emission was measured between 510 and 560 nm. Endogenous NAD(P) Cell Culture H was excited at 363 nm and emitted light was collected by using a INS-1E cells were cultured in RPMI 1640 medium containing 11 mM glucose 450–490 nm filter. Data acquisition and processing were performed by the supplemented with 10 mM HEPES (pH 7.3), 10% (v/v) heat-inactivated fetal MetaFluor software. NAD(P)H was monitored in cells superfused with H295R calf serum (Brunschwig AG; Switzerland), 50 mM b-mercaptoethanol, 1 mM superfusion medium at 30C.

Cell Metabolism 13, 601–611, May 4, 2011 ª2011 Elsevier Inc. 609 Cell Metabolism Signaling Function of Matrix Ca2+

Mitochondrial Membrane Potential Measurements Received: June 8, 2010 JC-1 fluorescence was measured ratiometrically at 37C in a multiwell fluores- Revised: December 30, 2010 cence reader (FlexStation, Molecular Devices Corporation; USA) (Park et al., Accepted: March 11, 2011 2008). Published: May 3, 2011

Oxygen Consumption REFERENCES INS-1E cells were plated onto polyornithine coated 24-well plates (Seahorse Biosciences) and infected as described above. Prior to the experiment, the Akhmedov, D., Braun, M., Mataki, C., Park, K.S., Pozzan, T., Schoonjans, K., cells were washed twice with KRBH containing 2.5 mM glucose. The plates Rorsman, P., Wollheim, C.B., and Wiederkehr, A. (2010). Mitochondrial matrix were then placed in the Seahorse instrument XF24 and oxygen consumption pH controls oxidative phosphorylation and metabolism-secretion coupling in rates were determined every 9 min. Stock solutions of glucose were added INS-1E clonal beta cells. FASEB J. 24, 4613–4626. during the run and immediately mixed to reach 16.7 mM. Babcock, D.F., Herrington, J., Goodwin, P.C., Park, Y.B., and Hille, B. (1997). 136 Immunocytochemistry Mitochondrial participation in the intracellular Ca2+ network. J. Cell Biol. , 833–844. H295R cells expressing mitoS100G were loaded with 200 nM MitoTracker Deep Red for 30 min in H295R superfusion medium and then fixed with 4% Balaban, R.S. (2009). The role of Ca(2+) signaling in the coordination of paraformaldehyde in PBS at room temperature for 15 min. The cells were mitochondrial ATP production with cardiac work. Biochim. Biophys. Acta rinsed several times with PBS + 100 mM glycine and permeabilized with 1787, 1334–1341. PBS + 1% BSA + 0.1% Triton X-100 for 15 min at room temperature. Blocking Bezprozvanny, I., Watras, J., and Ehrlich, B.E. (1991). Bell-shaped calcium- (1 hr) and antibody incubations were carried out in PBS + 5% BSA + 0.1% response curves of Ins(1,4,5)P3- and calcium-gated channels from endo- Triton X-100. Cells were incubated with a-S100G antibody (1:4000) for 1 hr. plasmic reticulum of cerebellum. Nature 351, 751–754. Goat anti-rabbit (H + L)-Alexa568 (1:1000) was used as secondary antibody Boerries, M., Most, P., Gledhill, J.R., Walker, J.E., Katus, H.A., Koch, W.J., for 1 hr. MitoTracker Deep Red was excited at 633 nm and the emitted Aebi, U., and Schoenenberger, C.A. (2007). Ca2+ -dependent interaction of fluorescent light was collected by using a 650 nm long-pass filter. INS-1E cells S100A1 with F1-ATPase leads to an increased ATP content in cardiomyo- expressing mitoS100G or mitoS100A1 were stained following a similar cytes. Mol. Cell. Biol. 27, 4365–4373. protocol. Capponi, A.M., Rossier, M.F., Davies, E., and Vallotton, M.B. (1988). Calcium Insulin Secretion stimulates steroidogenesis in permeabilized bovine adrenal cortical cells. 263 INS-1E cells were plated into polyornithine-coated 24-well tissue culture J. Biol. Chem. , 16113–16117. plates (Becton Dickinson; 400,000 cells/well). The cells were infected with Ca´ rdenas, C., Miller, R.A., Smith, I., Bui, T., Molgo´ , J., Mu¨ ller, M., Vais, H., adenoviruses as indicated in the figures and induced with DOX (1 mg/ml) for Cheung, K.H., Yang, J., Parker, I., et al. (2010). Essential regulation of cell 24 hr. Prior to the experiments, the cells were washed three times with bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria. KRBH containing 2.5mM glucose and 0.1% BSA. The cells were preincubated Cell 142, 270–283. for 30 min at 37C and then washed twice in the same buffer. Static secretion  Chalmers, S., and Nicholls, D.G. (2003). The relationship between free and experiments were performed for 30 min at 37 C in the presence of basal or total calcium concentrations in the matrix of liver and brain mitochondria. stimulatory glucose concentrations. Supernatants were saved for insulin J. Biol. Chem. 278, 19062–19070. measurements. For the determination of the insulin content, cells were ex- tracted in acid ethanol overnight at 4C. Insulin was measured by using an Cherradi, N., and Capponi, A.M. (1998). The acute regulation of mineralocorti- enzyme immunoassay kit (SPI-Bio, Montigny, France). coid biosynthesis: scenarios for the StAR system. Trends Endocrinol. Metab. 9, 412–418. Aldosterone Production Duchen, M.R. (1992). Ca(2+)-dependent changes in the mitochondrial ener- For the examination of aldosterone production 3.5 3 105 cells/well were getics in single dissociated mouse sensory neurons. Biochem. J. 283, 41–50. plated in a 24-well culture dish. The next day cells were infected as described Duchen, M.R. (2004). Mitochondria in health and disease: perspectives on above. Two days later, after a 2 hr preincubation in a medium devoid of a new mitochondrial biology. Mol. Aspects Med. 25, 365–451. UltroSer G, the cells were incubated for 2 hr (in a CO thermostat) in a similar 2 Gunter, T.E., and Sheu, S.S. (2009). Characteristics and possible functions of medium for hormone analysis. Aldosterone in the supernatant was determined mitochondrial Ca(2+) transport mechanisms. Biochim. Biophys. Acta 1787, with Coat-A-Count RIA kit (Siemens Healthcare Diagnostics, Deerfield, 1291–1308. IL, USA). Hajno´ czky, G., Robb-Gaspers, L.D., Seitz, M.B., and Thomas, A.P. (1995). 82 Statistical Analysis Decoding of cytosolic calcium oscillations in the mitochondria. Cell , For estimating significance of differences Student’s t test (Figures 2–6)or 415–424. factorial ANOVA and Tukey’s post-hoc test were used (Figure 7). Hajno´ czky, G., Csorda´ s, G., Das, S., Garcia-Perez, C., Saotome, M., Sinha Roy, S., and Yi, M. (2006). Mitochondrial calcium signalling and cell death: SUPPLEMENTAL INFORMATION approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis. Cell Calcium 40, 553–560. Supplemental Information includes three figures and can be found with this Hellman, B., Idahl, L.A., Lernmark, A., Sehlin, J., and Ta¨ ljedal, I.B. (1974). The article online at doi:10.1016/j.cmet.2011.03.015. pancreatic beta-cell recognition of insulin secretagogues. Effects of calcium and sodium on glucose metabolism and insulin release. Biochem. J. 138, ACKNOWLEDGMENTS 33–45. Henquin, J.C. (2009). Regulation of insulin secretion: a matter of phase control We thank Nicole Aebischer, Dale Brighouse, Olivier Dupont, Eszter Hala´ sz, and amplitude modulation. Diabetologia 52, 739–751. Elodie Husi, Danielle Nappey, and Aniko´ Rajki for expert technical assistance and Sergei Starchik for help with the analysis of the YC3.6 data. We gratefully Ishihara, H., Maechler, P., Gjinovci, A., Herrera, P.L., and Wollheim, C.B. acknowledge the continued support by the Swiss National Foundation (2003). Islet beta-cell secretion determines glucagon release from neighbour- 5 (310000-116750/1), the Ecole Polytechnique Fe´ de´ rale de Lausanne, EuroDia ing alpha-cells. Nat. Cell Biol. , 330–335. (LSHM-CT-2006-518153), a European Community-funded project under Ivarsson, R., Quintens, R., Dejonghe, S., Tsukamoto, K., in ’t Veld, P., Framework Program 6, and the Hungarian Council for Medical Research Renstro¨ m, E., and Schuit, F.C. (2005). Redox control of exocytosis: regulatory (ETT 008–09). role of NADPH, thioredoxin, and glutaredoxin. Diabetes 54, 2132–2142.

610 Cell Metabolism 13, 601–611, May 4, 2011 ª2011 Elsevier Inc. Cell Metabolism Signaling Function of Matrix Ca2+

Joseph, J.W., Jensen, M.V., Ilkayeva, O., Palmieri, F., Ala´ rcon, C., Rhodes, mitochondrial fission/fusion on metabolism-secretion coupling in C.J., and Newgard, C.B. (2006). The mitochondrial citrate/isocitrate carrier insulin-releasing cells. J. Biol. Chem. 283, 33347–33356. plays a regulatory role in glucose-stimulated insulin secretion. J. Biol. Chem. Perocchi, F., Gohil, V.M., Girgis, H.S., Bao, X.R., McCombs, J.E., Palmer, A.E., 281 , 35624–35632. and Mootha, V.K. (2010). MICU1 encodes a mitochondrial EF hand protein Jouaville, L.S., Pinton, P., Bastianutto, C., Rutter, G.A., and Rizzuto, R. (1999). required for Ca(2+) uptake. Nature 467, 291–296. Regulation of mitochondrial ATP synthesis by calcium: evidence for a long- Pralong, W.F., Bartley, C., and Wollheim, C.B. (1990). Single islet beta-cell 96 term metabolic priming. Proc. Natl. Acad. Sci. USA , 13807–13812. stimulation by nutrients: relationship between pyridine nucleotides, cytosolic Kennedy, E.D., Rizzuto, R., Theler, J.M., Pralong, W.F., Bastianutto, C., Ca2+ and secretion. EMBO J. 9, 53–60. Pozzan, T., and Wollheim, C.B. (1996). Glucose-stimulated insulin secretion Pralong, W.F., Hunyady, L., Va´ rnai, P., Wollheim, C.B., and Spa¨ t, A. (1992). correlates with changes in mitochondrial and cytosolic Ca2+ in aequorin-ex- Pyridine nucleotide redox state parallels production of aldosterone in potas- 98 pressing INS-1 cells. J. Clin. Invest. , 2524–2538. sium-stimulated adrenal glomerulosa cells. Proc. Natl. Acad. Sci. USA 89, Kennedy, E.D., Maechler, P., and Wollheim, C.B. (1998). Effects of depletion of 132–136. mitochondrial DNA in metabolism secretion coupling in INS-1 cells. Diabetes Pralong, W.F., Spa¨ t, A., and Wollheim, C.B. (1994). Dynamic pacing of cell 47, 374–380. metabolism by intracellular Ca2+ transients. J. Biol. Chem. 269, 27310–27314. Kennedy, H.J., Pouli, A.E., Ainscow, E.K., Jouaville, L.S., Rizzuto, R., and Ravier, M.A., Cheng-Xue, R., Palmer, A.E., Henquin, J.C., and Gilon, P. (2010). Rutter, G.A. (1999). Glucose generates sub-plasma membrane ATP microdo- Subplasmalemmal Ca(2+) measurements in mouse pancreatic beta cells mains in single islet beta-cells. Potential role for strategically located mito- support the existence of an amplifying effect of glucose on insulin secretion. chondria. J. Biol. Chem. 274, 13281–13291. Diabetologia 53, 1947–1957. Kirkpatrick, C.L., Marchetti, P., Purrello, F., Piro, S., Bugliani, M., Bosco, D., de Rizzuto, R., and Pozzan, T. (2006). Microdomains of intracellular Ca2+: molec- Koning, E.J., Engelse, M.A., Kerr-Conte, J., Pattou, F., and Wollheim, C.B. ular determinants and functional consequences. Physiol. Rev. 86, 369–408. (2010). Type 2 diabetes susceptibility expression in normal or diabetic sorted human alpha and beta cells: correlations with age or BMI of islet donors. Rizzuto, R., Brini, M., Murgia, M., and Pozzan, T. (1993). Microdomains with PLoS ONE 5, e11053. high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria. Science 262, 744–747. Linse, S., Johansson, C., Brodin, P., Grundstro¨ m, T., Drakenberg, T., and Forse´ n, S. (1991). Electrostatic contributions to the binding of Ca2+ in calbin- Rizzuto, R., Bastianutto, C., Brini, M., Murgia, M., and Pozzan, T. (1994). 126 din D9k. Biochemistry 30, 154–162. Mitochondrial Ca2+ homeostasis in intact cells. J. Cell Biol. , 1183–1194. Luciani, D.S., Misler, S., and Polonsky, K.S. (2006). Ca2+ controls slow NAD(P) Roha´ cs, T., Nagy, G., and Spa¨ t, A. (1997). Cytoplasmic Ca2+ signalling and H oscillations in glucose-stimulated mouse pancreatic islets. J. Physiol. 572, reduction of mitochondrial pyridine nucleotides in adrenal glomerulosa cells 322 379–392. in response to K+, angiotensin II and vasopressin. Biochem. J. , 785–792. Maechler, P., and Wollheim, C.B. (1999). Mitochondrial glutamate acts as Spa¨ t, A., and Hunyady, L. (2004). Control of aldosterone secretion: a model for a messenger in glucose-induced insulin exocytosis. Nature 402, 685–689. convergence in cellular signaling pathways. Physiol. Rev. 84, 489–539. Maechler, P., Kennedy, E.D., Wang, H., and Wollheim, C.B. (1998). Szanda, G., Koncz, P., Va´ rnai, P., and Spa¨ t, A. (2006). Mitochondrial Ca2+ Desensitization of mitochondrial Ca2+ and insulin secretion responses in the uptake with and without the formation of high-Ca2+ microdomains. Cell beta cell. J. Biol. Chem. 273, 20770–20778. Calcium 40, 527–537. Marenholz, I., Heizmann, C.W., and Fritz, G. (2004). S100 proteins in mouse Szanda, G., Koncz, P., Rajki, A., and Spa¨ t, A. (2008). Participation of p38 and man: from evolution to function and pathology (including an update of MAPK and a novel-type protein C in the control of mitochondrial the nomenclature). Biochem. Biophys. Res. Commun. 322, 1111–1122. Ca2+ uptake. Cell Calcium 43, 250–259. Ma´ rmol, P., Pardo, B., Wiederkehr, A., del Arco, A., Wollheim, C.B., and Territo, P.R., Mootha, V.K., French, S.A., and Balaban, R.S. (2000). Ca(2+) acti- Satru´ stegui, J. (2009). Requirement for aralar and its Ca2+-binding sites in vation of heart mitochondrial oxidative phosphorylation: role of the F(0)/F(1)- Ca2+ in mitochondria from INS-1 clonal beta-cells. ATPase. Am. J. Physiol. Cell Physiol. 278, C423–C435. J. Biol. Chem. 284, 515–524. Wiederkehr, A., and Wollheim, C.B. (2008). Impact of mitochondrial calcium on McCormack, J.G., Halestrap, A.P., and Denton, R.M. (1990). Role of calcium the coupling of metabolism to insulin secretion in the pancreatic beta-cell. Cell ions in regulation of mammalian intramitochondrial metabolism. Physiol. Calcium 44, 64–76. Rev. 70, 391–425. Wiederkehr, A., Park, K.S., Dupont, O., Demaurex, N., Pozzan, T., Cline, G.W., Park, K.S., Wiederkehr, A., Kirkpatrick, C., Mattenberger, Y., Martinou, J.C., and Wollheim, C.B. (2009). Matrix alkalinization: a novel mitochondrial signal Marchetti, P., Demaurex, N., and Wollheim, C.B. (2008). Selective actions of for sustained pancreatic beta-cell activation. EMBO J. 28, 417–428.

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