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The Mitochondrial Citrate/Isocitrate Carrier Plays a Regulatory Role in Glucose-stimulated Insulin Secretion *

The Mitochondrial Citrate/Isocitrate Carrier Plays a Regulatory Role in Glucose-stimulated... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 47, pp. 35624 –35632, November 24, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. The Mitochondrial Citrate/Isocitrate Carrier Plays a Regulatory Role in Glucose-stimulated Insulin Secretion Received for publication, March 20, 2006, and in revised form, August 23, 2006 Published, JBC Papers in Press, September 25, 2006, DOI 10.1074/jbc.M602606200 ‡ ‡ ‡ § ¶ Jamie W. Joseph , Mette V. Jensen , Olga Ilkayeva , Ferdinando Palmieri , Cristina Ala´rcon , ¶ ‡1 Christopher J. Rhodes , and Christopher B. Newgard From the Sarah W. Stedman Nutrition and Metabolism Center and Departments of Pharmacology and Cancer Biology, Medicine, and Biochemistry, Duke University Medical Center, Durham, North Carolina 27704, the Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, and National Research Council Institute of Biomembranes and Bioenergetics, University of Bari, 70125 Bari, Italy, and the Department of Medicine, Diabetes Center and Endocrine Division, University of Chicago, Chicago, Illinois 60637 Glucose-stimulated insulin secretion (GSIS) is mediated in closure of K channels at the plasma membrane. K chan- ATP ATP part by glucose metabolism-driven increases in ATP/ADP ratio, nel closure results in membrane depolarization and activation but by-products of mitochondrial glucose metabolism also play of voltage-dependent Ca channels, increasing the concentra- 2 2 an important role. Here we investigate the role of the mitochon- tion of cytosolic Ca (1–4). Elevation of cytosolic Ca pro- drial citrate/isocitrate carrier (CIC) in regulation of GSIS. Inhi- motes exocytosis of insulin-containing secretory granules (5). bition of CIC activity in INS-1-derived 832/13 cells or primary However, fluctuations in cytosolic Ca are not the only signal, rat islets by the substrate analogue 1,2,3-benzenetricarboxylate because under conditions of clamped cytosolic Ca concen- (BTC) resulted in potent inhibition of GSIS, involving both first trations, glucose can still cause significant insulin secretion (6). and second phase secretion. A recombinant adenovirus contain- This suggests that glucose generates signals/second messen- ing a CIC-specific siRNA (Ad-siCIC) dose-dependently reduced gers that are distinct from ATP and membrane depolarization CIC expression in 832/13 cells and caused parallel inhibitory for regulation of insulin secretion (7, 8). Some of the suggested effects on citrate accumulation in the cytosol. Ad-siCIC treat- mitochondrial factors include glutamate, malonyl-CoA, long- ment did not affect glucose utilization, glucose oxidation, or chain acyl-CoAs (LC-CoA), and/or NADPH (7–17). ATP/ADP ratio but did inhibit glucose incorporation into fatty The production of malonyl-CoA, LC-CoA, and NADPH in acids and glucose-induced increases in NADPH/NADP ratio the cytosol depends on the export of mitochondrial metabo- relative to cells treated with a control siRNA virus (Ad-siCon- lites. NADPH can be produced via one of three pyruvate cycling trol). Ad-siCIC also inhibited GSIS in 832/13 cells, whereas pathways, the pyruvate/malate pathway, the pyruvate/citrate overexpression of CIC enhanced GSIS and raised cytosolic cit- pathway, or the pyruvate/isocitrate pathway, via cytosolic rate levels. In normal rat islets, Ad-siCIC treatment also sup- NADP -dependent isoforms of malic enzyme (used in the pressed CIC mRNA levels and inhibited GSIS. We conclude that pyruvate/malate and pyruvate/citrate pathways) or cytosolic, export of citrate and/or isocitrate from the mitochondria to the NADP -dependent isocitrate dehydrogenase (ICDc) (used in cytosol is an important step in control of GSIS. the pyruvate/isocitrate cycle) (18, 19). Citrate emanating from mitochondrial metabolism can also be cleaved by ATP-citrate lyase to produce malonyl-CoA and LC-CoA (19). We and oth- The mechanism of glucose-stimulated insulin secretion ers have previously established that anaplerotic metabolism of (GSIS) from pancreatic islet -cells is not completely under- pyruvate and pyruvate cycling flux are closely correlated with stood. One component of the signaling pathway involves glu- the capacity for glucose-stimulated insulin secretion in -cells cose-induced increases in cytosolic ATP/ADP ratio, leading to (9, 13, 18, 20, 21). Also supporting a key role for anaplerosis is the earlier finding that 40–50% of pyruvate that enters mito- chondrial pathways at high glucose does so via pyruvate carbox- * This work was supported by a Canadian Institute of Health research fellow- ship (to J. W. J.), National Institutes of Health Grants DK42583 and DK58398 ylase, the anaplerotic entry point (22–24). (to C. B. N.) and DK51610 (to C. J. R.), a grant from the Ministero Operation of pyruvate cycles and generation of stimulus/se- dell’Istruzione, Universita e Ricerca (to F. P.), and a sponsored research cretion coupling factors from these pathways requires efficient agreement with Takeda Pharmaceuticals (to C. B. N.). The costs of publica- tion of this article were defrayed in part by the payment of page charges. export of tri- and dicarboxylic acids from the mitochondria to This article must therefore be hereby marked “advertisement” in accord- the cytosol. Two proteins that mediate these activities are the ance with 18 U.S.C. Section 1734 solely to indicate this fact. dicarboxylate carrier, which primarily transports malate, and To whom correspondence should be addressed: Sarah W. Stedman Nutri- tion and Metabolism Center, Duke University Medical Center, Independ- the tricarboxylate or citrate/isocitrate carrier (CIC), which cat- ence Park Facility, 4321 Medical Park Dr., Suite 200, Durham, NC 27704. Tel.: alyzes an electroneutral exchange of one of three tricarboxylic 919-668-6059; Fax: 919-477-0632; E-mail: [email protected]. acids (citrate, isocitrate, or cis-aconitate) plus a proton, for The abbreviations used are: GSIS, glucose-stimulated insulin secretion; CIC, cit- rate carrier; LC-CoA, long-chain acyl-CoA; ICDc, cytosolic isocitrate dehydro- another tricarboxylate-H , a dicarboxylate (malate or succi- genase; BTC, 1,2,3-benzenetricarboxylate; siRNA, small interfering RNA; Bis- nate), or phosphoenolpyruvate (19). Mitochondrial CIC occu- Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MTS, pies a critical position in intermediary metabolism, serving as a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium. key carbon source for the fatty acid and sterol biosynthetic 35624 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 •NOVEMBER 24, 2006 This is an Open Access article under the CC BY license. CIC and Glucose-stimulated Insulin Secretion pathways and for cytosolic NADPH production. Moreover, Glucose Utilization—832/13 cells were cultured with recent reports indicate that mouse islets lack malic enzyme [5- H]glucose as tracer at 0.08 Ci/mol, and samples were pro- cessed for measurement of glucose utilization as previously activity (25) and that siRNA-mediated suppression of cytosolic described (32). NADP-dependent ICDc activity strongly impairs GSIS, pyru- vate cycling, and NADPH production (18), suggesting that CIC Glucose Oxidation and Glucose Incorporation into Fatty could play a particularly important role in regulation of insulin Acids—832/13 cells were cultured with [U- C]glucose (0.5 Ci/mol) for 2 h, after which samples were loaded into a trap secretion. In the current study, we have tested this hypothesis system containing 1 N NaOH loaded into adjacent wells. The and demonstrate that inhibition of CIC with the specific sub- trap system was closed, and then the wells with media were strate analogue 1,2,3-benzenetricarboxylate (BTC) or by injected with 70% perchloric acid. The trap was incubated on a siRNA-mediated suppression of its expression results in potent shaker at 125 rpm for 90 min. NaOH was transferred to scintil- inhibition of GSIS, whereas CIC overexpression stimulates lation vials containing UniScint BD scintillation fluid, mixed, GSIS. and then counted. Glucose incorporation into lipid was meas- ured as previously described (33). EXPERIMENTAL PROCEDURES ADP and ATP Determination—Cellular ATP and ADP con- Cell Lines—The cell line 832/13 (26), derived from INS-1 rat tent was determined at the end of the 2-h incubation period, as insulinoma cells (27), was used in these studies. Cells were cul- described (34, 35). tured, and insulin secretion assays were performed as previ- Mitochondrial Membrane Potential Measurements—Glu- ously described (26). cose-induced changes in mitochondrial membrane potential siRNA Duplexes and Construction of the Ad-siCIC and Ad- were quantified in 832/13 cells maintained at 37 °C, as previ- CIC Overexpression Recombinant Adenovirus—Four siRNA ously described (35). Cells were loaded with rhodamine 123 (2.6 duplexes were tested against the rat citrate/isocitrate carrier M Rh123 for 20 min) (Molecular Probes, Inc., Eugene, OR). (accession number L12016). Relative to the start codon, the first Fluorescence was excited at 490 nm and measured at 530 nm. nucleotides targeted by each duplex were as follows: 136 (CIC Images were captured and analyzed using Metamorph software 136; AGT CTT CAC GTA TTC GGT CTT), 233 (CIC 233; (Molecular Devices Corp., Downingtown, PA). Exposure time CCG AAT ACG TGA AGA CTC ATT), 682 (CIC 682; CCG was 0.2 s, and images were acquired at0.2 Hz. Cells were first TGA AGG TGA AAT TCA TTT), and 922 (CIC 922; GCT treated with low glucose (2.8 mM) for 5 min and then with high ACT GTA CTG AAG CAG GTT). A scrambled siRNA glucose (20 mM) for 10 min. Changes in fluorescence were sequence with no known gene homology (GAG ACC CTA TCC determined by comparison of the fluorescence at 20 mM glu- GTG ATT A) was used as a control. siRNA duplexes were intro- cose over the last 5 min of treatment with the average fluores- duced into 832/13 cells at 50% confluence by nucleofection, cence over the first 50 s of exposure to 2.8 mM glucose, with the using the AMAXA system and the manufacturer’s protocols latter value set as 100%. At the end of an experiment, 1 M (Gaithersburg, MD). Experiments were performed 3 days after carbonyl cyanide p-trifluoromethoxyphenylhydrazone was duplex transfection. added to assess cell viability and to test the ability of the cell The CIC136 and scrambled control siRNA sequences were loaded with Rh123 to respond to changes in the mitochondrial used to prepare recombinant adenoviruses by previously membrane potential induced by the chemical uncoupler. described methods (28, 29). Recombinant adenoviruses con- Measurement of Citrate by Gas Chromatography/Mass taining the rat CIC cDNA sequence (AdCMV-CIC) or the bac- Spectrometry—Total citrate levels were measured relative to an terial -galactosidase gene (AdCMV-GAL) were prepared as added H -citrate internal standard (IsoTec) by gas chromatog- previously described (30, 31). For both the siRNA and overex- raphy/mass spectrometry, as previously described (13). Cytoso- pression viruses, virus-containing medium was purified using a lic and mitochondrial citrate levels were measured by the same BD Biosciences Adeno-X Purification Kit (Clontech, Palo Alto, method, with the following modifications. At the end of the CA), and virus titer was estimated by measurement of absorb- secretion assay, cells were washed twice with phosphate-buff- ance at 260 nm. ered saline and then treated with saponin (80 g/ml) for 20 min Real Time PCR Analysis of CIC mRNA Expression—RNA was (30 million cells/ml) to selectively permeabilize the plasma isolated from 832/13 cells using the Qiagen RNeasy Mini Kit membrane of cells in suspension without causing cell death (36, and from primary rat islets using the Qiagen MicroRNA kit 37). The cells were then centrifuged at 1000  g for 1 min. (Qiagen Inc., Valencia, CA). RNA was reverse transcribed using Supernatant was collected, representing the cytosolic fraction, the iScript cDNA synthesis kit (Bio-Rad). CIC mRNA levels and the cell pellet, representing the mitochondrial fraction, was were detected by real time PCR as previously described (13, 18), resuspended in 650 lof0.1 N HCl, and each fraction was used using prevalidated CIC and 18 S RNA-specific fluorescent for citrate analysis by gas chromatography/mass spectrometry. probes obtained from Applied Biosystems (Foster City, CA). CIC Immunoblot—Cellular proteins were extracted with cell NADPH and NADP Assays—832/13 cells were pretreated lysis buffer (Cell Signaling) containing phenylmethylsulfonyl for2hin2.8mM glucose and then incubated for2hat either 2.8 fluoride (0.5 mM), leupeptin (10 g/ml), aprotinin (10 g/ml), or 16.7 mM glucose. Cells were harvested and then snap frozen and pepstatin (5 g/ml). Extracts (40 g) were resolved on 10% in dry ice/ethanol. The cell pellets were stored at 80 °C until Bis-Tris SDS-polyacrylamide gels and electrotransferred to assayed. NADP and NADPH levels were measured as nitrocellulose membranes (Invitrogen). CIC was detected with described (13, 15, 18). a rabbit antibody against CIC (1:1000) (38) followed by horse- NOVEMBER 24, 2006• VOLUME 281 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 35625 CIC and Glucose-stimulated Insulin Secretion radish peroxidase-conjugated anti-rabbit antibody (1:10,000) (Amersham Biosciences). -Tubulin was detected by immuno- blotting with a mouse antibody against -tubulin (1:4000) (Sigma) followed by horseradish peroxidase-conjugated anti- mouse antibody (1:15,000) (Amersham Biosciences). Protein bands were detected with the ECL Advance immunoblot detec- tion kit (Amersham Biosciences). Islet Isolation and Insulin Secretion—Islets were harvested from adult male Sprague-Dawley rats weighing 250 g, as pre- viously described (13, 18, 28). Islets were incubated overnight in RPMI 1640 medium containing 8 mM glucose and supple- mented with 10% fetal bovine serum, 10 mM HEPES, 2 mM glutamine, 1 mM sodium pyruvate, 20 units/ml penicillin, 20 g/ml streptomycin, and 0.05 g/ml amphotericin B (Invitro- gen). Insulin secretion was performed with 20 islets for each condition. Islets were washed and incubated for1hin KRBH- bovine serum albumin secretion buffer containing 4.38 mM KCl, 1.2 mM MgSO , 1.5 mM KH PO , 129 mM NaCl, 5 mM 4 2 4 NaHCO ,10mM NaHEPES (Sigma), 3.11 mM CaCl , 0.25% 3 2 bovine serum albumin (Sigma), and 2.8 mM glucose. Islets were then incubated for 2 h in secretion buffer containing 2.8 mM glucose or 16.7 mM glucose in the presence or absence of 30 mM KCl. Insulin secretion and insulin content were measured as previously described (35). Cells were treated with 1000 parti- cles/ml of Ad-siCIC or Ad-siControl adenoviruses for 3 days unless otherwise stated. Islet Perifusion—60 islets were loaded onto a Swinnex 13 col- umn containing a nylon filter (Millipore, Burlington, MA). The chamber was perifused with KRBH-bovine serum albumin buffer with or without the addition of 0.5 or 2 mM BTC and containing various secretagogues at a flow rate of 0.5 ml/min using a Gilson Minipuls 3 pump (France). The temperature was maintained at 37 °C using an eight line in-line solution heater (Warner Instru- ments, Hamden, CT). Islets were perifused at low glucose (2.8 mM) for 45 min prior to each experiment. The solution was gassed with 95% O ,5%CO to achieve a pH of 7.4 and maintained at 37 °C. 2 2 Statistics—Statistical significance was assessed by Student’s t FIGURE 1. The CIC inhibitor BTC inhibits insulin secretion in 832/13 cells. test or by one-way or two-way analysis of variance for repeated A, insulin secretion in response to low glucose (LG; 2.8 mM), high glucose (HG; measures followed by multiple Bonferroni comparisons. All 16.7 mM), or high glucose plus KCl (HG KCl; 16.7 mM glucose 30 mM KCl). B, BTC dose-dependently inhibits glucose-stimulated insulin secretion. C, the data are expressed as means  S.E. effect of BTC on U- C glucose incorporation into lipids. Data represent the mean  S.E. for 8 –12 independent experiments. For all panels,*, p  0.05; RESULTS **, p  0.01, HG control versus HG BTC-treated; #, p  0.01 HG  KCl control versus HG  KCl plus BTC. The CIC Inhibitor BTC Inhibits GSIS in 832/13 Cells—As a first step in evaluation of the potential role of CIC in regulation of GSIS, we used the substrate analogue 1,2,3-benzenetricarboxylate (BTC; versus 2571  134 microunits/g of DNA; p  0.05). None of Sigma) to inhibit its activity in robustly glucose-responsive INS-1- the aforementioned effects of BTC could be ascribed to cyto- derived 832/13 cells. 2.0 mM BTC significantly inhibited both glu- toxicity, since treatment of 832/13 cells for3hwith2mM BTC cose-stimulated (16.7 mM) and glucose  KCl-stimulated (16.7 had no effect on cell viability, as assessed either by the Tox- mM glucose  30 mM KCl) insulin secretion without affecting iLight cytotoxicity assay or the MTS mitochondrial dye method basal insulin secretion (Fig. 1A). The effects of BTC on the -fold (data not shown). response to glucose were dose-dependent (Fig. 1B). Treatment of The CIC Inhibitor BTC Inhibits GSIS in Isolated Rat Islets— cells with 2 mM BTC resulted in reduction of the -fold response of We next sought to determine whether BTC inhibits GSIS in GSIS from 6.5  1.0-fold in control (non-drug-treated) cells to primary rat islets. In static incubation experiments, the addi- 3.9 0.7-fold. As would be expected of an inhibitor of CIC and tion of 2 mM BTC to islets inhibited insulin secretion in mitochondrial citrate export, BTC significantly reduced the response to 16.7 mM glucose or 16.7 mM glucose  30 mM KCl incorporation of radiolabeled glucose into the organically compared with islets incubated in the absence of the drug (Fig. extractable lipid fraction (Fig. 1C). Insulin content was slightly 2A). To learn more about the effects of BTC on the phases of decreased in response to 2 mM BTC in 832/13 cells (3052 377 insulin secretion, we performed islet perifusion studies involv- 35626 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 •NOVEMBER 24, 2006 CIC and Glucose-stimulated Insulin Secretion FIGURE 2. The effect of BTC on insulin secretion in isolated rat islets. A, insulin secretion measured by static incubation (2 h); B, insulin secretion measured by islet perifusion. The islet perifusion studies were performed in the presence or absence of 0.5 or 2 mM BTC, added during stimulation with high glucose (HG; 16.7 mM). Data represent the mean  S.E. of 3– 6 experi- ments. ***, p 0.001 HG control versus HG BTC; ##, p 0.001 HG KCl control versus HG  KCl plus BTC. FIGURE 3. Effects of suppression of CIC expression via four different siRNA duplexes in 832/13 cells. Four distinct siRNA duplexes specific for CIC ing preperifusion with 2.8 mM glucose for 45 min, followed by (siCIC136, siCIC233, siCIC682, and siCIC922) or a control siRNA duplex with no 2.8 mM glucose for 15 min, 16.7 mM glucose plus or minus BTC known sequence homology (siControl) were introduced into 832/13 cells by (0.5 or 2 mM) for 30 min, and then 15 min with 16.7 mM glucose electroporation, followed by 48 h of tissue culture before beginning the experiment. A, measurement of CIC mRNA levels by real time PCR. B, glucose- 30 mM KCl in the absence of BTC. These experiments revealed stimulated insulin secretion. Data represent the mean  S.E. for 3– 6 inde- that BTC inhibited both the first and second phases of insulin pendent experiments. **, p 0.01; ***, p 0.001 siControl versus the various secretion in response to 16.7 mM glucose (Fig. 2B). siCIC duplexes. Transfection-based siRNA-mediated Suppression of CIC Expression Inhibits GSIS—In order to further investigate the for further experiments, “low” (100 particles/cell), which role of CIC in control of GSIS by an alternative, nonpharmaco- caused no significant decrease in CIC mRNA levels, and “high” logic approach, 832/13 cells were electroporated with four dif- (500 particles/cell), which caused a suppression of 75  3% ferent siRNA duplexes against CIC or a control, nonspecific relative to Ad-siControl-treated cells. The low dose of siRNA duplex (siControl). The four CIC-specific siRNA Ad-siCIC did not affect glucose- or glucose  KCl-stimulated duplexes reduced CIC mRNA levels by 50–77% and caused insulin secretion, whereas the high dose of Ad-siCIC inhibited impairment of insulin secretion in response to high glucose to both glucose-stimulated (47  2%) and glucose  KCl-stimu- degrees (25–49%) in proportion to their efficacy for knock- lated insulin secretion (45  2%) compared with cells treated down of CIC expression (Fig. 3, A and B). Treatment of 832/13 with either Ad-siControl or the low dose of Ad-siCIC (Fig. 4C). cells with CIC siRNA duplexes did not affect insulin content Importantly, insulin secretion in the presence of low glucose compared with siControl-treated cells (data not shown). 30 mM KCl was not different in Ad-siCIC compared with Adenovirus-mediated Delivery of a CIC-specific siRNA Inhib- Ad-siControl-treated cells (430  14 versus 412  19 its GSIS—In order to further investigate the effects of CIC microunits/mg of protein; control versus Ad-siCIC), indicating knockdown, we constructed a recombinant adenovirus (Ad- that suppression of CIC expression does not interfere with siCIC) containing the siRNA sequence corresponding to the nutrient-independent stimulation of insulin secretory granule most effective duplex siRNA against CIC, based on the data in exocytosis. Fig. 3A (duplex CIC 136). Treatment of 832/13 cells with Ad- CIC Suppression Leads to Decreased Cytosolic Citrate Levels— siCIC caused a virus dose-dependent decrease in CIC mRNA We next sought to confirm our findings of effective siRNA- levels compared with cells treated with a control adenovirus mediated knockdown of CIC mRNA and protein levels (Figs. 3 (Ad-siControl) (Fig. 4A). Treatment of 832/13 cells with Ad- and 4) via measurement of the functional activity of CIC in siCIC also resulted in a 53 8% reduction in CIC protein levels living cells. To this end, we measured total, cytosolic, and mito- compared with cells treated with Ad-siControl or Ad-Gal chondrial citrate levels in 832/13 cells incubated at high glucose (Fig. 4B). Based on these studies, two viral doses were selected and treated with either Ad-siCIC or Ad-siControl. Treatment NOVEMBER 24, 2006• VOLUME 281 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 35627 CIC and Glucose-stimulated Insulin Secretion FIGURE 5. Treatment of 832/13 cells with Ad-siCIC reduces total and cyto- solic citrate levels. A, total cellular citrate levels; B, citrate levels in cytosolic and mitochondrial cell fractions, prepared as described under “Experimental Procedures.” Data represent the mean  S.E. of 3–5 independent experi- ments. ***, p  0.001 HG Ad-siControl versus HG Ad-siCIC. FIGURE 4. Effects of adenovirus-mediated suppression of CIC expression on insulin secretion in 832/13 cells. A, dose-dependent effects of Ad-siCIC on CIC mRNA levels, measured by real time PCR. B, immunoblot analysis in extracts from cells treated with AdCMV-Gal (lane 1), Ad-siCIC (lane 2), or ured a wide array of metabolic variables. Treatment of AdCMV-CIC (lane 3). The blot shown is representative of three independent 832/13 cells with the high dose of Ad-siCIC adenovirus did experiments, in which Ad-siCIC treatment caused an average 53  8% sup- pression and AdCMV-CIC treatment caused a 10 2-fold increase in CIC pro- not affect the glycolytic rate (Fig. 6A) or glucose oxidation tein levels compared with AdCMV-GAL-treated control cells. C, dose-de- (Fig. 6B) relative to Ad-siControl treatment but did reduce pendent effects of Ad-siCIC on insulin secretion. For the secretion studies, the viral titers indicated by the words low and high in A were used, which caused the incorporation of radiolabeled glucose into fatty acids by no significant suppression of CIC expression or a 74.7  3% suppression, 34  5% (Fig. 6C). Neither ATP or ADP levels were signifi- respectively. The viral dose of the Ad-siControl was the same as that for the cantly altered by Ad-siCIC treatment, and glucose caused high dose of Ad-siCIC (500 particles/cell). For both panels, data represent the mean S.E. of 8 –10 independent experiments., p 0.01 LG Ad-siControl identical increases in ATP/ADP ratio in Ad-siCIC relative to versus LG Ad-siCIC; ***, p 0.001 HG Ad-siControl versus HG Ad-siCIC; ##, p Ad-siControl-treated cells (data not shown). Consistent 0.01 HG  KCl Ad-siControl versus HG  KCl Ad-siCIC. with the latter finding, glucose-stimulated hyperpolarization of the mitochondrial membrane potential was not changed with Ad-siCIC resulted in a 37  3% decrease in total cellular in Ad-siCIC compared with Ad-siControl-treated cells (Ad- citrate levels and a 54  2% decrease in cytosolic citrate con- siControl-treated cells, 78  10%; Ad-siCIC-treated cells, centration, with no change in citrate content of intact mito- 84  6% fluorescence at 20 mM glucose relative to fluores- chondria (Fig. 5, A and B). Cytosolic and mitochondrial citrate cence at 2.8 mM glucose set to 100% for both groups). Finally, levels were discriminated by selective permeabilization of the recent studies have suggested that pyruvate/citrate and plasma membrane with a low concentration of saponin (80 pyruvate/isocitrate cycling may be linked to GSIS via pro- g/ml) (36, 37). The validity of the assay is supported by our duction of NADPH in the ICDc reaction (18). Consistent finding of recovery of 90% of total citrate lyase activity and1% with this idea, Ad-siCIC treatment resulted in lowering of of total citrate synthase in the cytosolic fraction (data not NADPH and NADP levels at both low and high glucose shown). These data are consistent with a significant decrease in levels but with a larger effect on NADPH, such that the the expression and functional activity of CIC, the major conduit for citrate export from mitochondria. NADPH/NADP ratio at stimulatory glucose and the incre- Metabolic Effects of CIC Suppression—In an effort to gain ment in NADPH/NADP as glucose was raised from low to insight into potential metabolic mechanisms linking sup- high levels was significantly reduced in Ad-siCIC-treated pression of CIC expression to impairment of GSIS, we meas- compared with Ad-siControl-treated cells (Fig. 7, A and B). 35628 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 •NOVEMBER 24, 2006 CIC and Glucose-stimulated Insulin Secretion FIGURE 7. The effects of Ad-siCIC on glucose-stimulated alterations in NADPH, NADP , and NADPH/NADP ratio in 832/13 cells. A, NADPH and NADP levels in response to LG and HG. **, p 0.01, NADPH HG Ad-siControl versus NADPH HG Ad-siCIC. B, NADPH/NADP ratio. **, p 0.01, HG Ad-siCon- trol versus HG Ad-siCIC. Data represent the mean  S.E. of five independent assays. lated insulin secretion in isolated rat islets (Fig. 9). Ad-siCIC treatment did not affect islet insulin content relative to Ad- siControl-treated islets (data not shown). FIGURE 6. Metabolic effects of Ad-siCIC treatment in 832/13 cells. A, gly- colytic rate; B, glucose oxidation; C,U- C glucose incorporation into fatty DISCUSSION acids. Data represent the mean  S.E. of 3– 4 independent assays. ***, p 0.001, HG Ad-siControl versus HG Ad-siCIC. The studies summarized herein demonstrate that the mito- chondrial tricarboxylate or citrate/isocitrate carrier (CIC) plays an Overexpression of CIC Enhances GSIS and Raises Cytosolic important role in GSIS. Inhibition of CIC activity by the specific Citrate Levels—Treatment of 832/13 cells with a recombinant substrate analogue BTC resulted in inhibition of GSIS in 832/13 adenovirus containing the rat CIC cDNA (AdCMV-CIC) cells. BTC also inhibited first- and second-phase insulin secretion resulted in a 10  2-fold increase in CIC protein levels as com- in isolated rat islets. The findings obtained with a pharmacologic pared with cells treated with AdCMV-GAL (Fig. 4B). tool for suppression of CIC were confirmed by molecular AdCMV-CIC treatment had no significant effect on insulin approaches. Thus, delivery of siRNA constructs specific for CIC secretion at low glucose but caused a 60% increase in secretion either by duplex transfection or in the context of a recombinant at stimulatory glucose relative to AdCMV-GAL-treated cells adenovirus (Ad-siCIC) caused a clear decrease in CIC mRNA and (Fig. 8A). In addition, AdCMV-CIC treatment increased cyto- protein levels and impaired GSIS in 832/13 cells. Knockdown of solic citrate levels in 832/13 cells by 40% compared with CIC was without effect on glucose utilization, glucose oxidation, or AdCMV-Gal-treated cells (Fig. 8B). ATP/ADP ratio but did cause significant lowering of glucose-stim- Modulation of CIC Expression in Rat Islets Regulates GSIS— ulated citrate accumulation in the cytosol and glucose incorpora- Finally, we tested the effect of manipulation of CIC expression tion into lipids in these cells. Conversely, CIC overexpression on GSIS in primary rat islets, facilitated by use of a recombinant resulted in increased accumulation of cytosolic citrate and adenovirus that allows us to deliver the CIC siRNA construct to enhanced GSIS in 832/13 cells. Finally, adenovirus-mediated sup- such cells with high efficiency (28). Treatment of rat islets with pression of CIC expression in primary rat islets impaired GSIS. Ad-siCIC reduced CIC mRNA levels by 55  9% (p  0.001) These findings support the concept that the ability of glucose to compared with Ad-siControl-treated islets. This amount of stimulate an increase in cytosolic citrate or isocitrate levels plays an CIC knockdown resulted in a 44  3% inhibition of glucose- important role in control of GSIS, independent of changes in ATP/ stimulated and a 32  4% inhibition of glucose  KCl-stimu- ADP ratio. It should also be noted that the lack of effect of CIC NOVEMBER 24, 2006• VOLUME 281 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 35629 CIC and Glucose-stimulated Insulin Secretion glucose retains an ability to stimulate insulin secretion even when ATP-sensitive K channels are rendered unresponsive, strongly implying that metabolic signals other than ATP/ADP ratio are important for GSIS (4, 11, 12). Several by-products of mitochondrial metabolism of glucose have been invoked as candidates for mediating this ancillary signaling pathway, including glutamate, malonyl-CoA, LC-CoA, and NADPH (7–10, 14, 24, 39). The current study provides clear evidence that coupling factors derived from the metabolism of citrate or isocitrate deserve particular scrutiny. Technically, citrate or isocitrate can be converted to -keto- glutarate, which can in turn be converted to glutamate by glu- tamate dehydrogenase or transaminases. However, studies in several laboratories have failed to identify increases in gluta- mate levels during glucose stimulation of -cells (8, 9, 40). Moreover, glutamate probably serves as an insulin secreta- gogue via glutaminolysis or oxidation of glutamate in the TCA cycle (41). Producing glutamate from citrate or isocitrate would thus seem to be an inefficient and unlikely mechanism for explaining our results. The malonyl-CoA/LC-CoA model of GSIS holds that during glucose stimulation, pyruvate carboxylase-mediated anaplero- sis raises cytosolic citrate levels, which leads in turn to an increase in malonyl-CoA levels (16, 17). Malonyl-CoA is a potent inhibitor of carnitine palmitoyltransferase I (42), and this action could divert LC-CoA away from oxidation in the FIGURE 8. CIC overexpression virus increases GSIS and cytosolic citrate levels in 832/13 cells. A, glucose-stimulated insulin secretion. B, citrate levels mitochondria toward accumulation in the cytosol (16, 17). in cytosolic and mitochondrial cell fractions, prepared as described under Consistent with this model, treatment of -cells with glucose “Experimental Procedures.” Data represent the mean S.E. of 3–5 independ- ent experiments. **, p  0.01; ***, p  0.001 HG AdCMV-Gal versus HG causes a rapid rise in malonyl-CoA levels that precedes insulin AdCMV-CIC; #, p  0.05 HG AdCMV-Gal versus HG AdCMV-CIC (mitochon- secretion (16). Glucose stimulation also suppresses fatty acid drial citrate). oxidation, and the addition of LC-CoA stimulates insulin gran- ule exocytosis in permeabilized -cells (43). However, LC-CoA also stimulates K channel activity in patch-clamped -cells ATP (44, 45), an effect seemingly at odds with a role of LC-CoA as a glucose-derived stimulus/secretion coupling factor. Further- more, prevention of the glucose-induced rise in malonyl-CoA levels by overexpression of malonyl-CoA decarboxylase has no impact on GSIS (33, 46), a finding recently confirmed by the laboratory that developed the malonyl-CoA/LC-CoA hypothe- sis (47). Similarly, treatment of -cells with Triacsin C, an inhibitor of LC-CoA synthetase, does not impair glucose responsiveness (33, 46). In a modification of the original hypothesis, it has recently been suggested that malonyl-CoA/ LC-CoA might be important for the potentiating effect of fatty acids on GSIS, since experiments with triacsin C and malonyl- FIGURE 9. Ad-siCIC treatment of primary rat islets. Freshly harvested rat CoA decarboxylase overexpression diminished this action of islets were treated with Ad-siControl or Ad-siCIC for 2 h, and GSIS was meas- fatty acids in -cell lines and rat islets (47), although different ured 72 h after viral treatment. Data represent the mean  S.E. for four inde- pendent experiments. *, p  0.05; ***, p  0.001 HG Ad-siControl versus HG results were obtained by another laboratory with malonyl-CoA Ad-siCIC. decarboxylase (46). Overall, there is now a consistent lack of evidence for a direct role of malonyl-CoA in regulation of GSIS, knockdown on glycolytic flux or glucose oxidation argues against a whereas its potential role in lipid-mediated potentiation of nonspecific or global effect of CIC knockdown on -cell glucose GSIS remains an open question. Since lipids were not included metabolism, as does our finding of no change in cell viability using in our analysis of insulin secretion in the present study, it is the MTS assay, which provides an index of mitochondrial unlikely that the effect of CIC blockade to suppress GSIS is due function. to lowering of malonyl-CoA levels. To date, alteration in ATP/ADP ratio is the only universally Among the current candidates for the ancillary mitochondri- accepted pathway that links glucose metabolism to insulin ally derived signal for insulin secretion, a role for NADPH is secretion (12). However, it is clear from numerous studies that supported by several lines of evidence. First, pyruvate cycling 35630 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 •NOVEMBER 24, 2006 CIC and Glucose-stimulated Insulin Secretion 6. Gembal, M., Gilon, P., and Henquin, J. C. (1992) J. Clin. Invest. 89, activity is strongly correlated with the capacity for GSIS in a 1288–1295 panel of INS-1-derived cell lines, and NADPH is an expected 7. MacDonald, P. E., Joseph, J. W., and Rorsman, P. (2005) Philos. Trans. R. by-product of all of the proposed pyruvate cycling pathways (8, Soc. Lond. B Biol. Sci. 360, 2211–2225 9, 13, 18–21). Second, the NADPH/NADP ratio increases in 8. MacDonald, M. J., Fahien, L. A., Brown, L. J., Hasan, N. M., Buss, J. D., and direct proportion to media glucose concentration and GSIS in Kendrick, M. A. (2005) Am. J. Physiol. 288, E1–E15 rodent islets and several -cell lines, whereas this relationship 9. Lu, D., Mulder, H., Zhao, P., Burgess, S. C., Jensen, M. V., Kamzolova, S., Newgard, C. B., and Sherry, A. D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, does not exist for NADH/NAD ratio and GSIS (15, 18). Third, 2708–2713 the addition of NADPH to patch-clamped -cells stimulated 10. Deeney, J. T., Prentki, M., and Corkey, B. E. (2000) Semin. Cell Dev. Biol. exocytosis as measured by increases in cell capacitance, 11, 267–275 whereas NADH had no effect. These studies also suggest that 11. Newgard, C. B., and McGarry, J. D. (1995) Annu. Rev. Biochem. 64, NADPH/NADP ratio may be the relevant signal, since the 689–719 addition of NADP reversed the stimulatory effect obtained 12. Newgard, C. B., and Matchinsky, F. M. (2001) in Handbook of Physiology Section 7: The Endocrine System (Jefferson, A. C., and Cherrington, A., with NADPH alone (15). Finally, suppression of ICDc expres- eds) Vol. II, pp. 125–151, Oxford University Press, Oxford sion by adenovirus-mediated delivery of a siRNA construct 13. Jensen, M. V., Joseph, J. W., Ilkayeva, O., Burgess, S., Lu, D., Ronnebaum, caused a coordinate reduction in pyruvate cycling activity and S. M., Odegaard, M., Becker, T. C., Sherry, A. D., and Newgard, C. B. NAPDH/NADP ratio and simultaneously caused strong (2006) J. Biol. Chem. 281, 22342–22351 impairment of GSIS in -cell lines and rat islets (18). The find- 14. Maechler, P., and Wollheim, C. B. (1999) Nature 402, 685–689 15. Ivarsson, R., Quintens, R., Dejonghe, S., Tsukamoto, K., in’t Veld, P., ings of the current study fully support the NADPH/NADP Renstrom, E., and Schuit, F. C. (2005) Diabetes 54, 2132–2142 ratio model, since we found that siRNA-mediated knockdown 16. Corkey, B. E., Glennon, M. C., Chen, K. S., Deeney, J. T., Matschinsky, of CIC caused significant decreases in NADPH levels as well as F. M., and Prentki, M. (1989) J. Biol. Chem. 264, 21608–21612 a reduction in the increment in NADPH/NADP ratio as glu- 17. Prentki, M., Vischer, S., Glennon, M. C., Regazzi, R., Deeney, J. T., and cose was raised from low to high levels. Corkey, B. E. (1992) J. Biol. Chem. 267, 5802–5810 If NADPH is an important signaling molecule for insulin 18. Ronnebaum, S. M., Ilkayeva, O., Burgess, S. C., Joseph, J. W., Lu, D., Stevens, R. D., Becker, T. C., Sherry, A. D., Newgard, C. B., and Jensen, secretion, the targets by which it mediates its effects remain to M. V. (2006) J. Biol. Chem. 281, 30593–30602 be identified (8). One interesting candidate appears to be volt- 19. Palmieri, F. (2004) Pflugers Arch. Eur. J. Physiol. 447, 689–709 age-gated K channels, which were recently shown to be regu- 20. Boucher, A., Lu, D., Burgess, S. C., Telemaque-Potts, S., Jensen, M. V., lated by changes in NADPH/NADP levels (48). However, it Mulder, H., Wang, M. Y., Unger, R. H., Sherry, A. D., and Newgard, C. B. also remains possible that non-NADPH-related byproducts of (2004) J. Biol. Chem. 279, 27263–27271 citrate and/or isocitrate metabolism in the cytosol are the crit- 21. Cline, G. W., Lepine, R. L., Papas, K. K., Kibbey, R. G., and Shulman, G. I. (2004) J. Biol. Chem. 279, 44370–44375 ical pyruvate cycling-related factors that regulate insulin secre- 22. MacDonald, M. J. (1993) Arch. Biochem. Biophys. 305, 205–214 tion. 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Physiol. 289, E218–E224 35632 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 •NOVEMBER 24, 2006 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

The Mitochondrial Citrate/Isocitrate Carrier Plays a Regulatory Role in Glucose-stimulated Insulin Secretion *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2006 Elsevier Inc.
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0021-9258
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10.1074/jbc.m602606200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 47, pp. 35624 –35632, November 24, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. The Mitochondrial Citrate/Isocitrate Carrier Plays a Regulatory Role in Glucose-stimulated Insulin Secretion Received for publication, March 20, 2006, and in revised form, August 23, 2006 Published, JBC Papers in Press, September 25, 2006, DOI 10.1074/jbc.M602606200 ‡ ‡ ‡ § ¶ Jamie W. Joseph , Mette V. Jensen , Olga Ilkayeva , Ferdinando Palmieri , Cristina Ala´rcon , ¶ ‡1 Christopher J. Rhodes , and Christopher B. Newgard From the Sarah W. Stedman Nutrition and Metabolism Center and Departments of Pharmacology and Cancer Biology, Medicine, and Biochemistry, Duke University Medical Center, Durham, North Carolina 27704, the Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, and National Research Council Institute of Biomembranes and Bioenergetics, University of Bari, 70125 Bari, Italy, and the Department of Medicine, Diabetes Center and Endocrine Division, University of Chicago, Chicago, Illinois 60637 Glucose-stimulated insulin secretion (GSIS) is mediated in closure of K channels at the plasma membrane. K chan- ATP ATP part by glucose metabolism-driven increases in ATP/ADP ratio, nel closure results in membrane depolarization and activation but by-products of mitochondrial glucose metabolism also play of voltage-dependent Ca channels, increasing the concentra- 2 2 an important role. Here we investigate the role of the mitochon- tion of cytosolic Ca (1–4). Elevation of cytosolic Ca pro- drial citrate/isocitrate carrier (CIC) in regulation of GSIS. Inhi- motes exocytosis of insulin-containing secretory granules (5). bition of CIC activity in INS-1-derived 832/13 cells or primary However, fluctuations in cytosolic Ca are not the only signal, rat islets by the substrate analogue 1,2,3-benzenetricarboxylate because under conditions of clamped cytosolic Ca concen- (BTC) resulted in potent inhibition of GSIS, involving both first trations, glucose can still cause significant insulin secretion (6). and second phase secretion. A recombinant adenovirus contain- This suggests that glucose generates signals/second messen- ing a CIC-specific siRNA (Ad-siCIC) dose-dependently reduced gers that are distinct from ATP and membrane depolarization CIC expression in 832/13 cells and caused parallel inhibitory for regulation of insulin secretion (7, 8). Some of the suggested effects on citrate accumulation in the cytosol. Ad-siCIC treat- mitochondrial factors include glutamate, malonyl-CoA, long- ment did not affect glucose utilization, glucose oxidation, or chain acyl-CoAs (LC-CoA), and/or NADPH (7–17). ATP/ADP ratio but did inhibit glucose incorporation into fatty The production of malonyl-CoA, LC-CoA, and NADPH in acids and glucose-induced increases in NADPH/NADP ratio the cytosol depends on the export of mitochondrial metabo- relative to cells treated with a control siRNA virus (Ad-siCon- lites. NADPH can be produced via one of three pyruvate cycling trol). Ad-siCIC also inhibited GSIS in 832/13 cells, whereas pathways, the pyruvate/malate pathway, the pyruvate/citrate overexpression of CIC enhanced GSIS and raised cytosolic cit- pathway, or the pyruvate/isocitrate pathway, via cytosolic rate levels. In normal rat islets, Ad-siCIC treatment also sup- NADP -dependent isoforms of malic enzyme (used in the pressed CIC mRNA levels and inhibited GSIS. We conclude that pyruvate/malate and pyruvate/citrate pathways) or cytosolic, export of citrate and/or isocitrate from the mitochondria to the NADP -dependent isocitrate dehydrogenase (ICDc) (used in cytosol is an important step in control of GSIS. the pyruvate/isocitrate cycle) (18, 19). Citrate emanating from mitochondrial metabolism can also be cleaved by ATP-citrate lyase to produce malonyl-CoA and LC-CoA (19). We and oth- The mechanism of glucose-stimulated insulin secretion ers have previously established that anaplerotic metabolism of (GSIS) from pancreatic islet -cells is not completely under- pyruvate and pyruvate cycling flux are closely correlated with stood. One component of the signaling pathway involves glu- the capacity for glucose-stimulated insulin secretion in -cells cose-induced increases in cytosolic ATP/ADP ratio, leading to (9, 13, 18, 20, 21). Also supporting a key role for anaplerosis is the earlier finding that 40–50% of pyruvate that enters mito- chondrial pathways at high glucose does so via pyruvate carbox- * This work was supported by a Canadian Institute of Health research fellow- ship (to J. W. J.), National Institutes of Health Grants DK42583 and DK58398 ylase, the anaplerotic entry point (22–24). (to C. B. N.) and DK51610 (to C. J. R.), a grant from the Ministero Operation of pyruvate cycles and generation of stimulus/se- dell’Istruzione, Universita e Ricerca (to F. P.), and a sponsored research cretion coupling factors from these pathways requires efficient agreement with Takeda Pharmaceuticals (to C. B. N.). The costs of publica- tion of this article were defrayed in part by the payment of page charges. export of tri- and dicarboxylic acids from the mitochondria to This article must therefore be hereby marked “advertisement” in accord- the cytosol. Two proteins that mediate these activities are the ance with 18 U.S.C. Section 1734 solely to indicate this fact. dicarboxylate carrier, which primarily transports malate, and To whom correspondence should be addressed: Sarah W. Stedman Nutri- tion and Metabolism Center, Duke University Medical Center, Independ- the tricarboxylate or citrate/isocitrate carrier (CIC), which cat- ence Park Facility, 4321 Medical Park Dr., Suite 200, Durham, NC 27704. Tel.: alyzes an electroneutral exchange of one of three tricarboxylic 919-668-6059; Fax: 919-477-0632; E-mail: [email protected]. acids (citrate, isocitrate, or cis-aconitate) plus a proton, for The abbreviations used are: GSIS, glucose-stimulated insulin secretion; CIC, cit- rate carrier; LC-CoA, long-chain acyl-CoA; ICDc, cytosolic isocitrate dehydro- another tricarboxylate-H , a dicarboxylate (malate or succi- genase; BTC, 1,2,3-benzenetricarboxylate; siRNA, small interfering RNA; Bis- nate), or phosphoenolpyruvate (19). Mitochondrial CIC occu- Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MTS, pies a critical position in intermediary metabolism, serving as a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium. key carbon source for the fatty acid and sterol biosynthetic 35624 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 •NOVEMBER 24, 2006 This is an Open Access article under the CC BY license. CIC and Glucose-stimulated Insulin Secretion pathways and for cytosolic NADPH production. Moreover, Glucose Utilization—832/13 cells were cultured with recent reports indicate that mouse islets lack malic enzyme [5- H]glucose as tracer at 0.08 Ci/mol, and samples were pro- cessed for measurement of glucose utilization as previously activity (25) and that siRNA-mediated suppression of cytosolic described (32). NADP-dependent ICDc activity strongly impairs GSIS, pyru- vate cycling, and NADPH production (18), suggesting that CIC Glucose Oxidation and Glucose Incorporation into Fatty could play a particularly important role in regulation of insulin Acids—832/13 cells were cultured with [U- C]glucose (0.5 Ci/mol) for 2 h, after which samples were loaded into a trap secretion. In the current study, we have tested this hypothesis system containing 1 N NaOH loaded into adjacent wells. The and demonstrate that inhibition of CIC with the specific sub- trap system was closed, and then the wells with media were strate analogue 1,2,3-benzenetricarboxylate (BTC) or by injected with 70% perchloric acid. The trap was incubated on a siRNA-mediated suppression of its expression results in potent shaker at 125 rpm for 90 min. NaOH was transferred to scintil- inhibition of GSIS, whereas CIC overexpression stimulates lation vials containing UniScint BD scintillation fluid, mixed, GSIS. and then counted. Glucose incorporation into lipid was meas- ured as previously described (33). EXPERIMENTAL PROCEDURES ADP and ATP Determination—Cellular ATP and ADP con- Cell Lines—The cell line 832/13 (26), derived from INS-1 rat tent was determined at the end of the 2-h incubation period, as insulinoma cells (27), was used in these studies. Cells were cul- described (34, 35). tured, and insulin secretion assays were performed as previ- Mitochondrial Membrane Potential Measurements—Glu- ously described (26). cose-induced changes in mitochondrial membrane potential siRNA Duplexes and Construction of the Ad-siCIC and Ad- were quantified in 832/13 cells maintained at 37 °C, as previ- CIC Overexpression Recombinant Adenovirus—Four siRNA ously described (35). Cells were loaded with rhodamine 123 (2.6 duplexes were tested against the rat citrate/isocitrate carrier M Rh123 for 20 min) (Molecular Probes, Inc., Eugene, OR). (accession number L12016). Relative to the start codon, the first Fluorescence was excited at 490 nm and measured at 530 nm. nucleotides targeted by each duplex were as follows: 136 (CIC Images were captured and analyzed using Metamorph software 136; AGT CTT CAC GTA TTC GGT CTT), 233 (CIC 233; (Molecular Devices Corp., Downingtown, PA). Exposure time CCG AAT ACG TGA AGA CTC ATT), 682 (CIC 682; CCG was 0.2 s, and images were acquired at0.2 Hz. Cells were first TGA AGG TGA AAT TCA TTT), and 922 (CIC 922; GCT treated with low glucose (2.8 mM) for 5 min and then with high ACT GTA CTG AAG CAG GTT). A scrambled siRNA glucose (20 mM) for 10 min. Changes in fluorescence were sequence with no known gene homology (GAG ACC CTA TCC determined by comparison of the fluorescence at 20 mM glu- GTG ATT A) was used as a control. siRNA duplexes were intro- cose over the last 5 min of treatment with the average fluores- duced into 832/13 cells at 50% confluence by nucleofection, cence over the first 50 s of exposure to 2.8 mM glucose, with the using the AMAXA system and the manufacturer’s protocols latter value set as 100%. At the end of an experiment, 1 M (Gaithersburg, MD). Experiments were performed 3 days after carbonyl cyanide p-trifluoromethoxyphenylhydrazone was duplex transfection. added to assess cell viability and to test the ability of the cell The CIC136 and scrambled control siRNA sequences were loaded with Rh123 to respond to changes in the mitochondrial used to prepare recombinant adenoviruses by previously membrane potential induced by the chemical uncoupler. described methods (28, 29). Recombinant adenoviruses con- Measurement of Citrate by Gas Chromatography/Mass taining the rat CIC cDNA sequence (AdCMV-CIC) or the bac- Spectrometry—Total citrate levels were measured relative to an terial -galactosidase gene (AdCMV-GAL) were prepared as added H -citrate internal standard (IsoTec) by gas chromatog- previously described (30, 31). For both the siRNA and overex- raphy/mass spectrometry, as previously described (13). Cytoso- pression viruses, virus-containing medium was purified using a lic and mitochondrial citrate levels were measured by the same BD Biosciences Adeno-X Purification Kit (Clontech, Palo Alto, method, with the following modifications. At the end of the CA), and virus titer was estimated by measurement of absorb- secretion assay, cells were washed twice with phosphate-buff- ance at 260 nm. ered saline and then treated with saponin (80 g/ml) for 20 min Real Time PCR Analysis of CIC mRNA Expression—RNA was (30 million cells/ml) to selectively permeabilize the plasma isolated from 832/13 cells using the Qiagen RNeasy Mini Kit membrane of cells in suspension without causing cell death (36, and from primary rat islets using the Qiagen MicroRNA kit 37). The cells were then centrifuged at 1000  g for 1 min. (Qiagen Inc., Valencia, CA). RNA was reverse transcribed using Supernatant was collected, representing the cytosolic fraction, the iScript cDNA synthesis kit (Bio-Rad). CIC mRNA levels and the cell pellet, representing the mitochondrial fraction, was were detected by real time PCR as previously described (13, 18), resuspended in 650 lof0.1 N HCl, and each fraction was used using prevalidated CIC and 18 S RNA-specific fluorescent for citrate analysis by gas chromatography/mass spectrometry. probes obtained from Applied Biosystems (Foster City, CA). CIC Immunoblot—Cellular proteins were extracted with cell NADPH and NADP Assays—832/13 cells were pretreated lysis buffer (Cell Signaling) containing phenylmethylsulfonyl for2hin2.8mM glucose and then incubated for2hat either 2.8 fluoride (0.5 mM), leupeptin (10 g/ml), aprotinin (10 g/ml), or 16.7 mM glucose. Cells were harvested and then snap frozen and pepstatin (5 g/ml). Extracts (40 g) were resolved on 10% in dry ice/ethanol. The cell pellets were stored at 80 °C until Bis-Tris SDS-polyacrylamide gels and electrotransferred to assayed. NADP and NADPH levels were measured as nitrocellulose membranes (Invitrogen). CIC was detected with described (13, 15, 18). a rabbit antibody against CIC (1:1000) (38) followed by horse- NOVEMBER 24, 2006• VOLUME 281 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 35625 CIC and Glucose-stimulated Insulin Secretion radish peroxidase-conjugated anti-rabbit antibody (1:10,000) (Amersham Biosciences). -Tubulin was detected by immuno- blotting with a mouse antibody against -tubulin (1:4000) (Sigma) followed by horseradish peroxidase-conjugated anti- mouse antibody (1:15,000) (Amersham Biosciences). Protein bands were detected with the ECL Advance immunoblot detec- tion kit (Amersham Biosciences). Islet Isolation and Insulin Secretion—Islets were harvested from adult male Sprague-Dawley rats weighing 250 g, as pre- viously described (13, 18, 28). Islets were incubated overnight in RPMI 1640 medium containing 8 mM glucose and supple- mented with 10% fetal bovine serum, 10 mM HEPES, 2 mM glutamine, 1 mM sodium pyruvate, 20 units/ml penicillin, 20 g/ml streptomycin, and 0.05 g/ml amphotericin B (Invitro- gen). Insulin secretion was performed with 20 islets for each condition. Islets were washed and incubated for1hin KRBH- bovine serum albumin secretion buffer containing 4.38 mM KCl, 1.2 mM MgSO , 1.5 mM KH PO , 129 mM NaCl, 5 mM 4 2 4 NaHCO ,10mM NaHEPES (Sigma), 3.11 mM CaCl , 0.25% 3 2 bovine serum albumin (Sigma), and 2.8 mM glucose. Islets were then incubated for 2 h in secretion buffer containing 2.8 mM glucose or 16.7 mM glucose in the presence or absence of 30 mM KCl. Insulin secretion and insulin content were measured as previously described (35). Cells were treated with 1000 parti- cles/ml of Ad-siCIC or Ad-siControl adenoviruses for 3 days unless otherwise stated. Islet Perifusion—60 islets were loaded onto a Swinnex 13 col- umn containing a nylon filter (Millipore, Burlington, MA). The chamber was perifused with KRBH-bovine serum albumin buffer with or without the addition of 0.5 or 2 mM BTC and containing various secretagogues at a flow rate of 0.5 ml/min using a Gilson Minipuls 3 pump (France). The temperature was maintained at 37 °C using an eight line in-line solution heater (Warner Instru- ments, Hamden, CT). Islets were perifused at low glucose (2.8 mM) for 45 min prior to each experiment. The solution was gassed with 95% O ,5%CO to achieve a pH of 7.4 and maintained at 37 °C. 2 2 Statistics—Statistical significance was assessed by Student’s t FIGURE 1. The CIC inhibitor BTC inhibits insulin secretion in 832/13 cells. test or by one-way or two-way analysis of variance for repeated A, insulin secretion in response to low glucose (LG; 2.8 mM), high glucose (HG; measures followed by multiple Bonferroni comparisons. All 16.7 mM), or high glucose plus KCl (HG KCl; 16.7 mM glucose 30 mM KCl). B, BTC dose-dependently inhibits glucose-stimulated insulin secretion. C, the data are expressed as means  S.E. effect of BTC on U- C glucose incorporation into lipids. Data represent the mean  S.E. for 8 –12 independent experiments. For all panels,*, p  0.05; RESULTS **, p  0.01, HG control versus HG BTC-treated; #, p  0.01 HG  KCl control versus HG  KCl plus BTC. The CIC Inhibitor BTC Inhibits GSIS in 832/13 Cells—As a first step in evaluation of the potential role of CIC in regulation of GSIS, we used the substrate analogue 1,2,3-benzenetricarboxylate (BTC; versus 2571  134 microunits/g of DNA; p  0.05). None of Sigma) to inhibit its activity in robustly glucose-responsive INS-1- the aforementioned effects of BTC could be ascribed to cyto- derived 832/13 cells. 2.0 mM BTC significantly inhibited both glu- toxicity, since treatment of 832/13 cells for3hwith2mM BTC cose-stimulated (16.7 mM) and glucose  KCl-stimulated (16.7 had no effect on cell viability, as assessed either by the Tox- mM glucose  30 mM KCl) insulin secretion without affecting iLight cytotoxicity assay or the MTS mitochondrial dye method basal insulin secretion (Fig. 1A). The effects of BTC on the -fold (data not shown). response to glucose were dose-dependent (Fig. 1B). Treatment of The CIC Inhibitor BTC Inhibits GSIS in Isolated Rat Islets— cells with 2 mM BTC resulted in reduction of the -fold response of We next sought to determine whether BTC inhibits GSIS in GSIS from 6.5  1.0-fold in control (non-drug-treated) cells to primary rat islets. In static incubation experiments, the addi- 3.9 0.7-fold. As would be expected of an inhibitor of CIC and tion of 2 mM BTC to islets inhibited insulin secretion in mitochondrial citrate export, BTC significantly reduced the response to 16.7 mM glucose or 16.7 mM glucose  30 mM KCl incorporation of radiolabeled glucose into the organically compared with islets incubated in the absence of the drug (Fig. extractable lipid fraction (Fig. 1C). Insulin content was slightly 2A). To learn more about the effects of BTC on the phases of decreased in response to 2 mM BTC in 832/13 cells (3052 377 insulin secretion, we performed islet perifusion studies involv- 35626 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 •NOVEMBER 24, 2006 CIC and Glucose-stimulated Insulin Secretion FIGURE 2. The effect of BTC on insulin secretion in isolated rat islets. A, insulin secretion measured by static incubation (2 h); B, insulin secretion measured by islet perifusion. The islet perifusion studies were performed in the presence or absence of 0.5 or 2 mM BTC, added during stimulation with high glucose (HG; 16.7 mM). Data represent the mean  S.E. of 3– 6 experi- ments. ***, p 0.001 HG control versus HG BTC; ##, p 0.001 HG KCl control versus HG  KCl plus BTC. FIGURE 3. Effects of suppression of CIC expression via four different siRNA duplexes in 832/13 cells. Four distinct siRNA duplexes specific for CIC ing preperifusion with 2.8 mM glucose for 45 min, followed by (siCIC136, siCIC233, siCIC682, and siCIC922) or a control siRNA duplex with no 2.8 mM glucose for 15 min, 16.7 mM glucose plus or minus BTC known sequence homology (siControl) were introduced into 832/13 cells by (0.5 or 2 mM) for 30 min, and then 15 min with 16.7 mM glucose electroporation, followed by 48 h of tissue culture before beginning the experiment. A, measurement of CIC mRNA levels by real time PCR. B, glucose- 30 mM KCl in the absence of BTC. These experiments revealed stimulated insulin secretion. Data represent the mean  S.E. for 3– 6 inde- that BTC inhibited both the first and second phases of insulin pendent experiments. **, p 0.01; ***, p 0.001 siControl versus the various secretion in response to 16.7 mM glucose (Fig. 2B). siCIC duplexes. Transfection-based siRNA-mediated Suppression of CIC Expression Inhibits GSIS—In order to further investigate the for further experiments, “low” (100 particles/cell), which role of CIC in control of GSIS by an alternative, nonpharmaco- caused no significant decrease in CIC mRNA levels, and “high” logic approach, 832/13 cells were electroporated with four dif- (500 particles/cell), which caused a suppression of 75  3% ferent siRNA duplexes against CIC or a control, nonspecific relative to Ad-siControl-treated cells. The low dose of siRNA duplex (siControl). The four CIC-specific siRNA Ad-siCIC did not affect glucose- or glucose  KCl-stimulated duplexes reduced CIC mRNA levels by 50–77% and caused insulin secretion, whereas the high dose of Ad-siCIC inhibited impairment of insulin secretion in response to high glucose to both glucose-stimulated (47  2%) and glucose  KCl-stimu- degrees (25–49%) in proportion to their efficacy for knock- lated insulin secretion (45  2%) compared with cells treated down of CIC expression (Fig. 3, A and B). Treatment of 832/13 with either Ad-siControl or the low dose of Ad-siCIC (Fig. 4C). cells with CIC siRNA duplexes did not affect insulin content Importantly, insulin secretion in the presence of low glucose compared with siControl-treated cells (data not shown). 30 mM KCl was not different in Ad-siCIC compared with Adenovirus-mediated Delivery of a CIC-specific siRNA Inhib- Ad-siControl-treated cells (430  14 versus 412  19 its GSIS—In order to further investigate the effects of CIC microunits/mg of protein; control versus Ad-siCIC), indicating knockdown, we constructed a recombinant adenovirus (Ad- that suppression of CIC expression does not interfere with siCIC) containing the siRNA sequence corresponding to the nutrient-independent stimulation of insulin secretory granule most effective duplex siRNA against CIC, based on the data in exocytosis. Fig. 3A (duplex CIC 136). Treatment of 832/13 cells with Ad- CIC Suppression Leads to Decreased Cytosolic Citrate Levels— siCIC caused a virus dose-dependent decrease in CIC mRNA We next sought to confirm our findings of effective siRNA- levels compared with cells treated with a control adenovirus mediated knockdown of CIC mRNA and protein levels (Figs. 3 (Ad-siControl) (Fig. 4A). Treatment of 832/13 cells with Ad- and 4) via measurement of the functional activity of CIC in siCIC also resulted in a 53 8% reduction in CIC protein levels living cells. To this end, we measured total, cytosolic, and mito- compared with cells treated with Ad-siControl or Ad-Gal chondrial citrate levels in 832/13 cells incubated at high glucose (Fig. 4B). Based on these studies, two viral doses were selected and treated with either Ad-siCIC or Ad-siControl. Treatment NOVEMBER 24, 2006• VOLUME 281 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 35627 CIC and Glucose-stimulated Insulin Secretion FIGURE 5. Treatment of 832/13 cells with Ad-siCIC reduces total and cyto- solic citrate levels. A, total cellular citrate levels; B, citrate levels in cytosolic and mitochondrial cell fractions, prepared as described under “Experimental Procedures.” Data represent the mean  S.E. of 3–5 independent experi- ments. ***, p  0.001 HG Ad-siControl versus HG Ad-siCIC. FIGURE 4. Effects of adenovirus-mediated suppression of CIC expression on insulin secretion in 832/13 cells. A, dose-dependent effects of Ad-siCIC on CIC mRNA levels, measured by real time PCR. B, immunoblot analysis in extracts from cells treated with AdCMV-Gal (lane 1), Ad-siCIC (lane 2), or ured a wide array of metabolic variables. Treatment of AdCMV-CIC (lane 3). The blot shown is representative of three independent 832/13 cells with the high dose of Ad-siCIC adenovirus did experiments, in which Ad-siCIC treatment caused an average 53  8% sup- pression and AdCMV-CIC treatment caused a 10 2-fold increase in CIC pro- not affect the glycolytic rate (Fig. 6A) or glucose oxidation tein levels compared with AdCMV-GAL-treated control cells. C, dose-de- (Fig. 6B) relative to Ad-siControl treatment but did reduce pendent effects of Ad-siCIC on insulin secretion. For the secretion studies, the viral titers indicated by the words low and high in A were used, which caused the incorporation of radiolabeled glucose into fatty acids by no significant suppression of CIC expression or a 74.7  3% suppression, 34  5% (Fig. 6C). Neither ATP or ADP levels were signifi- respectively. The viral dose of the Ad-siControl was the same as that for the cantly altered by Ad-siCIC treatment, and glucose caused high dose of Ad-siCIC (500 particles/cell). For both panels, data represent the mean S.E. of 8 –10 independent experiments., p 0.01 LG Ad-siControl identical increases in ATP/ADP ratio in Ad-siCIC relative to versus LG Ad-siCIC; ***, p 0.001 HG Ad-siControl versus HG Ad-siCIC; ##, p Ad-siControl-treated cells (data not shown). Consistent 0.01 HG  KCl Ad-siControl versus HG  KCl Ad-siCIC. with the latter finding, glucose-stimulated hyperpolarization of the mitochondrial membrane potential was not changed with Ad-siCIC resulted in a 37  3% decrease in total cellular in Ad-siCIC compared with Ad-siControl-treated cells (Ad- citrate levels and a 54  2% decrease in cytosolic citrate con- siControl-treated cells, 78  10%; Ad-siCIC-treated cells, centration, with no change in citrate content of intact mito- 84  6% fluorescence at 20 mM glucose relative to fluores- chondria (Fig. 5, A and B). Cytosolic and mitochondrial citrate cence at 2.8 mM glucose set to 100% for both groups). Finally, levels were discriminated by selective permeabilization of the recent studies have suggested that pyruvate/citrate and plasma membrane with a low concentration of saponin (80 pyruvate/isocitrate cycling may be linked to GSIS via pro- g/ml) (36, 37). The validity of the assay is supported by our duction of NADPH in the ICDc reaction (18). Consistent finding of recovery of 90% of total citrate lyase activity and1% with this idea, Ad-siCIC treatment resulted in lowering of of total citrate synthase in the cytosolic fraction (data not NADPH and NADP levels at both low and high glucose shown). These data are consistent with a significant decrease in levels but with a larger effect on NADPH, such that the the expression and functional activity of CIC, the major conduit for citrate export from mitochondria. NADPH/NADP ratio at stimulatory glucose and the incre- Metabolic Effects of CIC Suppression—In an effort to gain ment in NADPH/NADP as glucose was raised from low to insight into potential metabolic mechanisms linking sup- high levels was significantly reduced in Ad-siCIC-treated pression of CIC expression to impairment of GSIS, we meas- compared with Ad-siControl-treated cells (Fig. 7, A and B). 35628 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 •NOVEMBER 24, 2006 CIC and Glucose-stimulated Insulin Secretion FIGURE 7. The effects of Ad-siCIC on glucose-stimulated alterations in NADPH, NADP , and NADPH/NADP ratio in 832/13 cells. A, NADPH and NADP levels in response to LG and HG. **, p 0.01, NADPH HG Ad-siControl versus NADPH HG Ad-siCIC. B, NADPH/NADP ratio. **, p 0.01, HG Ad-siCon- trol versus HG Ad-siCIC. Data represent the mean  S.E. of five independent assays. lated insulin secretion in isolated rat islets (Fig. 9). Ad-siCIC treatment did not affect islet insulin content relative to Ad- siControl-treated islets (data not shown). FIGURE 6. Metabolic effects of Ad-siCIC treatment in 832/13 cells. A, gly- colytic rate; B, glucose oxidation; C,U- C glucose incorporation into fatty DISCUSSION acids. Data represent the mean  S.E. of 3– 4 independent assays. ***, p 0.001, HG Ad-siControl versus HG Ad-siCIC. The studies summarized herein demonstrate that the mito- chondrial tricarboxylate or citrate/isocitrate carrier (CIC) plays an Overexpression of CIC Enhances GSIS and Raises Cytosolic important role in GSIS. Inhibition of CIC activity by the specific Citrate Levels—Treatment of 832/13 cells with a recombinant substrate analogue BTC resulted in inhibition of GSIS in 832/13 adenovirus containing the rat CIC cDNA (AdCMV-CIC) cells. BTC also inhibited first- and second-phase insulin secretion resulted in a 10  2-fold increase in CIC protein levels as com- in isolated rat islets. The findings obtained with a pharmacologic pared with cells treated with AdCMV-GAL (Fig. 4B). tool for suppression of CIC were confirmed by molecular AdCMV-CIC treatment had no significant effect on insulin approaches. Thus, delivery of siRNA constructs specific for CIC secretion at low glucose but caused a 60% increase in secretion either by duplex transfection or in the context of a recombinant at stimulatory glucose relative to AdCMV-GAL-treated cells adenovirus (Ad-siCIC) caused a clear decrease in CIC mRNA and (Fig. 8A). In addition, AdCMV-CIC treatment increased cyto- protein levels and impaired GSIS in 832/13 cells. Knockdown of solic citrate levels in 832/13 cells by 40% compared with CIC was without effect on glucose utilization, glucose oxidation, or AdCMV-Gal-treated cells (Fig. 8B). ATP/ADP ratio but did cause significant lowering of glucose-stim- Modulation of CIC Expression in Rat Islets Regulates GSIS— ulated citrate accumulation in the cytosol and glucose incorpora- Finally, we tested the effect of manipulation of CIC expression tion into lipids in these cells. Conversely, CIC overexpression on GSIS in primary rat islets, facilitated by use of a recombinant resulted in increased accumulation of cytosolic citrate and adenovirus that allows us to deliver the CIC siRNA construct to enhanced GSIS in 832/13 cells. Finally, adenovirus-mediated sup- such cells with high efficiency (28). Treatment of rat islets with pression of CIC expression in primary rat islets impaired GSIS. Ad-siCIC reduced CIC mRNA levels by 55  9% (p  0.001) These findings support the concept that the ability of glucose to compared with Ad-siControl-treated islets. This amount of stimulate an increase in cytosolic citrate or isocitrate levels plays an CIC knockdown resulted in a 44  3% inhibition of glucose- important role in control of GSIS, independent of changes in ATP/ stimulated and a 32  4% inhibition of glucose  KCl-stimu- ADP ratio. It should also be noted that the lack of effect of CIC NOVEMBER 24, 2006• VOLUME 281 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 35629 CIC and Glucose-stimulated Insulin Secretion glucose retains an ability to stimulate insulin secretion even when ATP-sensitive K channels are rendered unresponsive, strongly implying that metabolic signals other than ATP/ADP ratio are important for GSIS (4, 11, 12). Several by-products of mitochondrial metabolism of glucose have been invoked as candidates for mediating this ancillary signaling pathway, including glutamate, malonyl-CoA, LC-CoA, and NADPH (7–10, 14, 24, 39). The current study provides clear evidence that coupling factors derived from the metabolism of citrate or isocitrate deserve particular scrutiny. Technically, citrate or isocitrate can be converted to -keto- glutarate, which can in turn be converted to glutamate by glu- tamate dehydrogenase or transaminases. However, studies in several laboratories have failed to identify increases in gluta- mate levels during glucose stimulation of -cells (8, 9, 40). Moreover, glutamate probably serves as an insulin secreta- gogue via glutaminolysis or oxidation of glutamate in the TCA cycle (41). Producing glutamate from citrate or isocitrate would thus seem to be an inefficient and unlikely mechanism for explaining our results. The malonyl-CoA/LC-CoA model of GSIS holds that during glucose stimulation, pyruvate carboxylase-mediated anaplero- sis raises cytosolic citrate levels, which leads in turn to an increase in malonyl-CoA levels (16, 17). Malonyl-CoA is a potent inhibitor of carnitine palmitoyltransferase I (42), and this action could divert LC-CoA away from oxidation in the FIGURE 8. CIC overexpression virus increases GSIS and cytosolic citrate levels in 832/13 cells. A, glucose-stimulated insulin secretion. B, citrate levels mitochondria toward accumulation in the cytosol (16, 17). in cytosolic and mitochondrial cell fractions, prepared as described under Consistent with this model, treatment of -cells with glucose “Experimental Procedures.” Data represent the mean S.E. of 3–5 independ- ent experiments. **, p  0.01; ***, p  0.001 HG AdCMV-Gal versus HG causes a rapid rise in malonyl-CoA levels that precedes insulin AdCMV-CIC; #, p  0.05 HG AdCMV-Gal versus HG AdCMV-CIC (mitochon- secretion (16). Glucose stimulation also suppresses fatty acid drial citrate). oxidation, and the addition of LC-CoA stimulates insulin gran- ule exocytosis in permeabilized -cells (43). However, LC-CoA also stimulates K channel activity in patch-clamped -cells ATP (44, 45), an effect seemingly at odds with a role of LC-CoA as a glucose-derived stimulus/secretion coupling factor. Further- more, prevention of the glucose-induced rise in malonyl-CoA levels by overexpression of malonyl-CoA decarboxylase has no impact on GSIS (33, 46), a finding recently confirmed by the laboratory that developed the malonyl-CoA/LC-CoA hypothe- sis (47). Similarly, treatment of -cells with Triacsin C, an inhibitor of LC-CoA synthetase, does not impair glucose responsiveness (33, 46). In a modification of the original hypothesis, it has recently been suggested that malonyl-CoA/ LC-CoA might be important for the potentiating effect of fatty acids on GSIS, since experiments with triacsin C and malonyl- FIGURE 9. Ad-siCIC treatment of primary rat islets. Freshly harvested rat CoA decarboxylase overexpression diminished this action of islets were treated with Ad-siControl or Ad-siCIC for 2 h, and GSIS was meas- fatty acids in -cell lines and rat islets (47), although different ured 72 h after viral treatment. Data represent the mean  S.E. for four inde- pendent experiments. *, p  0.05; ***, p  0.001 HG Ad-siControl versus HG results were obtained by another laboratory with malonyl-CoA Ad-siCIC. decarboxylase (46). Overall, there is now a consistent lack of evidence for a direct role of malonyl-CoA in regulation of GSIS, knockdown on glycolytic flux or glucose oxidation argues against a whereas its potential role in lipid-mediated potentiation of nonspecific or global effect of CIC knockdown on -cell glucose GSIS remains an open question. Since lipids were not included metabolism, as does our finding of no change in cell viability using in our analysis of insulin secretion in the present study, it is the MTS assay, which provides an index of mitochondrial unlikely that the effect of CIC blockade to suppress GSIS is due function. to lowering of malonyl-CoA levels. To date, alteration in ATP/ADP ratio is the only universally Among the current candidates for the ancillary mitochondri- accepted pathway that links glucose metabolism to insulin ally derived signal for insulin secretion, a role for NADPH is secretion (12). However, it is clear from numerous studies that supported by several lines of evidence. First, pyruvate cycling 35630 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 •NOVEMBER 24, 2006 CIC and Glucose-stimulated Insulin Secretion 6. Gembal, M., Gilon, P., and Henquin, J. C. (1992) J. Clin. 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Biol. exocytosis as measured by increases in cell capacitance, 11, 267–275 whereas NADH had no effect. These studies also suggest that 11. Newgard, C. B., and McGarry, J. D. (1995) Annu. Rev. Biochem. 64, NADPH/NADP ratio may be the relevant signal, since the 689–719 addition of NADP reversed the stimulatory effect obtained 12. Newgard, C. B., and Matchinsky, F. M. (2001) in Handbook of Physiology Section 7: The Endocrine System (Jefferson, A. C., and Cherrington, A., with NADPH alone (15). Finally, suppression of ICDc expres- eds) Vol. II, pp. 125–151, Oxford University Press, Oxford sion by adenovirus-mediated delivery of a siRNA construct 13. Jensen, M. V., Joseph, J. W., Ilkayeva, O., Burgess, S., Lu, D., Ronnebaum, caused a coordinate reduction in pyruvate cycling activity and S. M., Odegaard, M., Becker, T. C., Sherry, A. D., and Newgard, C. B. NAPDH/NADP ratio and simultaneously caused strong (2006) J. Biol. 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C., Sherry, A. D., Newgard, C. B., and Jensen, secretion, the targets by which it mediates its effects remain to M. V. (2006) J. Biol. Chem. 281, 30593–30602 be identified (8). One interesting candidate appears to be volt- 19. Palmieri, F. (2004) Pflugers Arch. Eur. J. Physiol. 447, 689–709 age-gated K channels, which were recently shown to be regu- 20. Boucher, A., Lu, D., Burgess, S. C., Telemaque-Potts, S., Jensen, M. V., lated by changes in NADPH/NADP levels (48). However, it Mulder, H., Wang, M. Y., Unger, R. H., Sherry, A. D., and Newgard, C. B. also remains possible that non-NADPH-related byproducts of (2004) J. Biol. Chem. 279, 27263–27271 citrate and/or isocitrate metabolism in the cytosol are the crit- 21. Cline, G. W., Lepine, R. L., Papas, K. K., Kibbey, R. G., and Shulman, G. I. (2004) J. Biol. Chem. 279, 44370–44375 ical pyruvate cycling-related factors that regulate insulin secre- 22. MacDonald, M. J. (1993) Arch. Biochem. Biophys. 305, 205–214 tion. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Nov 24, 2006

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