Title: Phosphoenolpyruvate Cycling via Mitochondrial Phosphoenolpyruvate Carboxykinase Links Anaplerosis and Mitochondrial GTP with Insulin Secretion
Abstract: Pancreatic β-cells couple the oxidation of glucose to the secretion of insulin. Apart from the canonical KATP-dependent glucose-stimulated insulin secretion (GSIS), there are important KATP-independent mechanisms involving both anaplerosis and mitochondrial GTP (mtGTP). How mtGTP that is trapped within the mitochondrial matrix regulates the cytosolic calcium increases that drive GSIS remains a mystery. Here we have investigated whether the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) is the GTPase linking hydrolysis of mtGTP made by succinyl-CoA synthetase (SCS-GTP) to an anaplerotic pathway producing phosphoenolpyruvate (PEP). Although cytosolic PEPCK (PEPCK-C) is absent, PEPCK-M message and protein were detected in INS-1 832/13 cells, rat islets, and mouse islets. PEPCK enzymatic activity is half that of primary hepatocytes and is localized exclusively to the mitochondria. Novel 13C-labeling strategies in INS-1 832/13 cells and islets measured substantial contribution of PEPCK-M to the synthesis of PEP. As high as 30% of PEP in INS-1 832/13 cells and 41% of PEP in rat islets came from PEPCK-M. The contribution of PEPCK-M to overall PEP synthesis more than tripled with glucose stimulation. Silencing the PEPCK-M gene completely inhibited GSIS underscoring its central role in mitochondrial metabolism-mediated insulin secretion. Given that mtGTP synthesized by SCS-GTP is an indicator of TCA flux that is crucial for GSIS, PEPCK-M is a strong candidate to link mtGTP synthesis with insulin release through anaplerotic PEP cycling. Pancreatic β-cells couple the oxidation of glucose to the secretion of insulin. Apart from the canonical KATP-dependent glucose-stimulated insulin secretion (GSIS), there are important KATP-independent mechanisms involving both anaplerosis and mitochondrial GTP (mtGTP). How mtGTP that is trapped within the mitochondrial matrix regulates the cytosolic calcium increases that drive GSIS remains a mystery. Here we have investigated whether the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) is the GTPase linking hydrolysis of mtGTP made by succinyl-CoA synthetase (SCS-GTP) to an anaplerotic pathway producing phosphoenolpyruvate (PEP). Although cytosolic PEPCK (PEPCK-C) is absent, PEPCK-M message and protein were detected in INS-1 832/13 cells, rat islets, and mouse islets. PEPCK enzymatic activity is half that of primary hepatocytes and is localized exclusively to the mitochondria. Novel 13C-labeling strategies in INS-1 832/13 cells and islets measured substantial contribution of PEPCK-M to the synthesis of PEP. As high as 30% of PEP in INS-1 832/13 cells and 41% of PEP in rat islets came from PEPCK-M. The contribution of PEPCK-M to overall PEP synthesis more than tripled with glucose stimulation. Silencing the PEPCK-M gene completely inhibited GSIS underscoring its central role in mitochondrial metabolism-mediated insulin secretion. Given that mtGTP synthesized by SCS-GTP is an indicator of TCA flux that is crucial for GSIS, PEPCK-M is a strong candidate to link mtGTP synthesis with insulin release through anaplerotic PEP cycling. β-Cells in pancreatic islets of Langerhans make and release insulin in response to changes in blood glucose levels. The mechanisms by which high concentrations of glucose stimulate insulin release from islets remain unclear. The canonical explanation for GSIS 2The abbreviations used are: GSISglucose-stimulated insulin secretionOAAoxaloacetic acidPEPCKphosphoenolpyruvate (PEP) carboxykinasePEPCK-Mmitochondrial PEPCKPEPCK-CcytosolicPKpyruvate kinasePCpyruvate carboxylaseα-KGα-ketoglutarateSCSsuccinyl-CoA synthetasemtGTPmitochondrial GTP2-PG2-phosphoglycerate3-PG3-phosphoglycerateMRMmultiple reaction monitoringCICcitrate carriercIDHcytosolic isocitrate dehydrogenaseTCAtricarboxylic acidsiRNAsmall interfering RNAGAPDHglyceraldehyde-3-phosphate dehydrogenaseDMEMDulbecco's modified Eagle's mediumMRMmultiple reaction monitoringLC/MS/MSliquid chromatography-tandem mass spectroscopyAPEatomic percent excessANOVAanalysis of variancercfrelative centrifugal force. is that glucose metabolism increases mitochondrial ATP production, thereby raising the cytosolic ATP:ADP ratio that triggers the closure of ATP-sensitive K+ channels. This, in turn, depolarizes the membrane and stimulates the opening of voltage-dependent Ca2+ channels with increased Ca2+ influx promoting the exocytosis of insulin. Although KATP channels certainly have an important role in β-cells, KATP-independent signals are implicated to play a fundamental role in GSIS. In particular, β-cells are known to have notably elevated rates of anaplerotic flux of the carbon from glucose into the mitochondria and back out to pyruvate (pyruvate cycling) that is tightly correlated with insulin secretion (1Newgard C.B. Lu D. Jensen M.V. Schissler J. Boucher A. Burgess S. Sherry A.D. Diabetes. 2002; 51: S389-393Crossref PubMed Google Scholar, 2Lu D. Mulder H. Zhao P. Burgess S.C. Jensen M.V. Kamzolova S. Newgard C.B. Sherry A.D. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 2708-2713Crossref PubMed Scopus (221) Google Scholar, 3Pongratz R.L. Kibbey R.G. Shulman G.I. Cline G.W. J. Biol. Chem. 2007; 282: 200-207Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 4Cline G.W. Lepine R.L. Papas K.K. Kibbey R.G. Shulman G.I. J. Biol. Chem. 2004; 279: 44370-44375Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). glucose-stimulated insulin secretion oxaloacetic acid phosphoenolpyruvate (PEP) carboxykinase mitochondrial PEPCK cytosolic pyruvate kinase pyruvate carboxylase α-ketoglutarate succinyl-CoA synthetase mitochondrial GTP 2-phosphoglycerate 3-phosphoglycerate multiple reaction monitoring citrate carrier cytosolic isocitrate dehydrogenase tricarboxylic acid small interfering RNA glyceraldehyde-3-phosphate dehydrogenase Dulbecco's modified Eagle's medium multiple reaction monitoring liquid chromatography-tandem mass spectroscopy atomic percent excess analysis of variance relative centrifugal force. Recently, mtGTP synthesis was identified as a novel KATP-independent mitochondrial signal for insulin secretion (5Kibbey R.G. Pongratz R.L. Romanelli A.J. Wollheim C.B. Cline G.W. Shulman G.I. Cell Metab. 2007; 5: 253-264Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). mtGTP is synthesized as a product of glucose metabolism by the GTP-specific isoform of the matrix enzyme SCS. mtGTP synthetic rates are determined by the rate of TCA cycle flux as well as by the ratio of activities of the ATP-specific and GTP-specific isoforms of SCS. The mtGTP signal is trapped within the matrix of the mitochondria, suggesting that another GTPase in the matrix transmits the mtGTP signal to the cytosol. Because both mtGTP synthesis and anaplerotic flux correlate with insulin secretion, we investigated whether the GTP-dependent mitochondrial isoform of PEPCK, an enzyme that lies at the intersection of anaplerosis and mtGTP metabolism (see Fig. 1A), is important for GSIS. INS-1 832/13 cells were cultured and siRNA-transfected, GSIS assays were performed, and islets were isolated as previously described (5Kibbey R.G. Pongratz R.L. Romanelli A.J. Wollheim C.B. Cline G.W. Shulman G.I. Cell Metab. 2007; 5: 253-264Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Primary hepatocytes were isolated by standard procedures from Sprague-Dawley rats (300g) from the Yale University Liver Center. As a control, a non-silencing siRNA 5′-AATTCTCCGAACGTGTCACGT-3′ (Qiagen) was used. Mitochondrial PEPCK (PEPCK-M) was targeted by two different siRNAs (Qiagen) with the following DNA templates: #1, 5′-AACGTGAACAATTTGACATTA-3′; #2, 5′-TCCCATTGGGCTCGTACCAAA-3′. Quantitation of mRNA by reverse transcription and real-time PCR was performed using the following primers: cytosolic PEPCK (PEPCK-C) (5′-ATGACACCCTCCTCCTGCAT-3′, 5′-CAGGAAGTGAGGAAGTTTGTGG-3′), PEPCK-M (5′-TTATGCACGATCCCTTTGCCATGC-3′, 5′-TCCTTCCTTTGGTACGAGCCCAAT-3′), and GAPDH (5′-GTTACCAGGGCTGCCTTCTC-3′, 5′-GGGTTTCCCGTTGATGACC-3′) as well as β-actin and SCS-GTP as previously described (5Kibbey R.G. Pongratz R.L. Romanelli A.J. Wollheim C.B. Cline G.W. Shulman G.I. Cell Metab. 2007; 5: 253-264Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Proteins were detected by Western blot analysis after separating 30–50 μg of total protein lysate on a 4–12% Tris-glycine gel (Invitrogen) and transferring to a polyvinylidene difluoride membrane (Immobilon-P 0.45 μm, Millipore). The sheep polyclonal PEPCK-C antibody was a kind gift from Daryl Granner (Vanderbilt University Medical Center). Goat anti-PEPCK-M (Abcam) and rabbit anti-GAPDH (Abcam) were used to compare PEPCK-M versus PEPCK-C in cultured cells, islets, and liver. A rabbit PEPCK-M antibody (Santa Cruz) was used in knockdown experiments. Cells or islets were homogenized in 1 ml of ice-cold isolation buffer (10 mm Hepes, pH 7.4, 250 mm sucrose, 1 mm EDTA, and 1 mm dithiothreitol) using a Potter-Elvehjem Teflon pestle by 50 vertical passes on ice. The supernatant from a 5-min 2000 rcf spin of the lysates was used for whole cell assays. Mitochondria were isolated from this supernatant by centrifugation at 10,000 rcf for 10 min. The pellet was solubilized in 0.4% deoxycholate on ice for 20 min before the insoluble material was pelleted by a second spin at 16,100 rcf for 10 min. To determine the percent mitochondrial activity, mitochondria were isolated from 1 ml of the post-nuclear supernatant as described above and dissolved in 100 μl of 0.4% deoxycholate on ice for 20 min, and 900 μl of the isolation buffer was then added for a final volume of 1 ml. 4% deoxycholate was added to the cytosol-containing supernatant from the mitochondrial spin to a final concentration of 0.04%. Cytosolic and mitochondrial fractions were assayed simultaneously. PEPCK activities were measured in the direction of oxaloacetate (OAA) formation as previously described with some modifications (6Wiese T.J. Lambeth D.O. Ray P.D. Comp. Biochem. Physiol. B. 1991; 100: 297-302Crossref PubMed Scopus (23) Google Scholar). The reaction was coupled to malate dehydrogenase for detection of NADH oxidation by fluorescence. Deoxy-GDP (dGDP) was used as a reactant in place of GDP or IDP because it discriminates against pyruvate kinase (PK), which is known to be high in insulin-secreting tissues (7Petrescu I. Bojan O. Saied M. Bârzu O. Schmidt F. Kühnle H.F. Anal. Biochem. 1979; 96: 279-281Crossref PubMed Scopus (102) Google Scholar). The reactions were performed in quadruplicate in 96-well plates with a 200-μl final volume containing 110 mm imidazole-Cl, pH 6.8, 3 mm MgSO4, 3 mm MnCl2, 13 mm NaF, 10 mm phenylalanine, 1 μm rotenone, 30 mm NaHCO3, 0.15 mm NADH, 6 units/ml malate dehydrogenase, 2 mm PEP, 0.5 mm dGDP, and cell homogenate containing ∼5–50 μg of protein. Magnesium was left out when comparing cytosolic to mitochondrial activities because it favors PEPCK-C. Control samples were run simultaneously in the absence of HCO3−/CO2, and this background slope was subtracted from the slope of the complete reaction. Before the experiment, the reaction mixture was freshly gassed with 100% CO2 for 10 min. The reaction was initiated with 0.5 mm dGDP, and the drop in NADH signal was assayed at 37 °C for 10–20 min at 10-s intervals by fluorescence using 335 nm for excitation and 460 nm for emission (Flex Station 3, Molecular Devices). Samples were assayed for glutamate dehydrogenase activity in the direction of glutamate formation from α-ketoglutarate (α-KG) in the presence of NH3 at 37 °C and pH 7.4 in a 250-μl total reaction mixture containing 70 mm triethanolamine, 3 mm EDTA, 125 mm ammonium acetate, 1.25 mm ADP, 0.25 mm NADH, 1.3 units/ml lactate dehydrogenase, and cell homogenate. The reactions were initiated with addition of 10 μl of 233 mm α-KG or water as a control and the decrease in absorption at 340 nm was monitored. Activities were obtained by subtracting the background slope of the samples without α-KG from those with α-KG. Mass isotopologue analysis of PEPCK activity was performed in INS-1 832/13 cells or rat islets preincubated in DMEM base without glucose for 1 h to minimize the amount of unlabeled glycolytic carbon in the cells. For the INS-1 832/13 cells, the medium was then aspirated and subsequently replaced with G0, G3, G7, or G15 mm unlabeled glucose containing either 5 mm unlabeled or [3-13C]pyruvate (Cambridge Isotope Laboratories. As such, [3-13C]pyruvate was used as a tracer entering distal to the irreversible PK step to label the mitochondrial OAA precursor pool of PEPCK-M and kept at a constant concentration with varying concentrations of glucose. To ensure that the [3-13C]pyruvate could contribute label to the PEP precursor pool in the face of increasing glucose concentrations, an extracellular concentration of 5 mm was applied. β-Cells in intact islets do not take up pyruvate but do take up glutamine, and even low glucose concentrations sufficiently increase mtGTP synthesis to prevent islet glutamine metabolism (via allosteric inhibition of glutamate dehydrogenase) (8Ishihara H. Wang H. Drewes L.R. Wollheim C.B. J. Clin. Invest. 1999; 104: 1621-1629Crossref PubMed Scopus (162) Google Scholar). Thus, after preincubation, the islets were incubated in either unlabeled or 1,2-13C-labeled glutamine in the absence of glucose. Parallel studies using unlabeled substrate were performed to account for natural abundance contribution to the signal. After substrate incubations, INS 832/13 cells that were cultured in 6-well plates were quenched by rapid wash with ice-cold water and then collected in 200 μl of ice-cold buffer A (5% acetonitrile, 2 mm ammonium acetate, 10 μm EDTA) with 10 μm d4-taurine (CDN Isotopes) as a load control (n = 6). For experiments with islets, after the incubation 100 islets per replicate were transferred to microcentrifuge tubes and centrifuged at 2000 rcf for 5 min to pellet the islets which were immediately frozen and lyophilized before resuspension in buffer A. The collected cells or islets were centrifuged to pellet bulk insoluble materials before filtration (MultiScreen BV, Millipore). The lysates were separated on a C16 5-μm 120 Å 4.6 × 250-mm column (Acclaim® Polar Advantage, Dionex) before ionization for multiple reaction monitoring (MRM) analysis by LC/MS/MS (Applied Biosystems MDS SCIEX, 4000 Q-TRAP). Each analyte was eluted isocratically in buffer A at a flow rate of 400 μl/min with a single gaussian-shaped peak whose retention time was confirmed with known standards. Individual MRM transition pairs (Q1/Q3) were designated for the natural abundance (M) and mass + 1 (M +1) isotopologues of each analyte (see Table 1). In addition to malate and PEP, aspartate and combined 2-phosphoglycerate/3-phosphoglycerate (2-PG/3-PG) enrichments were measured for comparison. Endogenous taurine and d4-taurine were used as internal and external controls. 13C incorporation into taurine did not occur under the experimental conditions nor did the absolute concentration of taurine change during the incubations. 2-PG and 3-PG were unable to be separated either chromatographically or by MRM analysis. So, for comparison with the atomic percent excess (APE) of PEP, they were treated as the same analyte for calculation of APEs because of equilibrium of both phosphoglycerate species across phosphoglycerate mutase. Phosphate was the daughter anion for both PEP and 2-PG/3-PG and sulfate for taurine as well as d4-taurine and did not vary with 13C incorporation. For the remaining analytes, because the daughter fragment contained two or more carbons, the M and M+1 of the daughter anions were summed. Under the conditions of these experiments, M + 2 and greater labeling were minimal. When multiple Q3 daughter anions were measurable for a single parent, the one with the highest signal to noise ratio and minimal spectral overlap were chosen and coincidence of both MRM pairs was confirmed in the chromatogram. The KEGG data base of metabolites was screened for potential significant M and M+1 mono- and di-anions that might give similar MRM pairs. Each analyte was determined to have either a unique MRM pair and/or retention time. Concentrations of each metabolite are presented relative to taurine calibrated by standard curves and referenced to an internal taurine concentration of 12.0 nmol/mg of protein (5Kibbey R.G. Pongratz R.L. Romanelli A.J. Wollheim C.B. Cline G.W. Shulman G.I. Cell Metab. 2007; 5: 253-264Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar).TABLE 1LC/MS/MS masses, retention times, and natural abundance measurementsCompoundMass/fragmentNatural abundanceRetention time% M+1minMalateM0133/714.45 ± 0.0273.89 ± 0.002M+1134/71, 134/72AspartateM0132/885.55 ± 0.2463.70 ± 0.093M+1133/88, 133/89PEPM0166.6/793.62 ± 0.083.85 ± 0.002M+1167.6/792-PG/3-PGM0185/793.52 ± 0.0893.85 ± 0.003M+1186/79Taurine123.6/805.52 ± 0.002d4-Taurine127.6/805.54 ± 0.002 Open table in a new tab The signal from natural abundance 13C in standards or in samples to which 13C-enriched substrates were not added (Table 1) was as predicted from 1.11% background enrichment (3.33% for PEP and 4.44% for malate) and did not change with increasing glucose concentration (supplemental Fig. S1). As such, the similarities of the natural abundance enrichments of the unlabeled samples with increasing glucose concentrations to the predicted values further supports that the peaks analyzed are the mono-anions of PEP and malate. At low concentrations, PEP had higher than predicted natural abundance from the decreased signal to noise ratio for M+1. Calculation of APEs was as traditionally defined, APE = 100 × (rsa − rbk)/((rsa − rbk) + 1), where r is the ratio of (M+1)/(M) for the sample, rsa, and background, rbk. Calculations of the contribution of PEPCK-M to relative glycolytic flux use the following definitions and assumptions (Fig. 1). 1) Glycolytic flux is defined as the contribution of unlabeled carbon into PEP in the glycolytic direction from enolase (νGlyc+1). 2) PEPCK-M is the only significant source of PEPCK activity in islets and INS-1 832/13 cells (νPEPCK-M+1 ≫ νPEPCK-C+1). 3) The only reactions contributing significant carbon to PEP synthesis are enolase (νGlyc+1) and PEPCK-M (νPEPCK-M+1), and there is negligible reverse flux from pyruvate kinase into PEP (νPK-1). 4) The only reaction contributing to significant disappearance of PEP is PK (i.e. there is minimal net reverse enolase (νGlyc-1)or PEPCK (νPEPCK-C-1 + νPEPCK-M-1) flux). 5) The size of the mitochondrial anion pools (i.e. of malate and PEP) are small relative to and in rapid communication with the cytosolic pool (9Garber A.J. Ballard F.J. J. Biol. Chem. 1969; 244: 4696-4703Abstract Full Text PDF PubMed Google Scholar). 6) PEP derived from PEPCK-M (PEPmt) and from enolase (PEPGlyc) is metabolically indistinguishable by PK. 7) Malate is primarily of mitochondrial origin and is in mass isotopologue equilibrium with OAA in both pools (10Wood H.G. Lifson N. Lorber V. J. Biol. Chem. 1945; 159: 475-489Abstract Full Text PDF Google Scholar, 11Shreeve W.W. De Meutter R.C. J. Biol. Chem. 1964; 239: 729-734Abstract Full Text PDF PubMed Google Scholar). Thus, PEP is treated as a single pool whose composition is determined by the rate of appearance of PEP (νGlyc+1 + νPEPCK-M+1) minus the rate of disappearance of PEP (νPK+1). Because νPK+1 for PEPmt and PEPGlyc is proportional to their population of this pool at any given time, the fractional contribution of PEPCK-M flux relative to glycolytic flux (ΦPEPCK-M) during the course of the incubations can be estimated to be ΦPEPCK-M = 100 × νPEPCK-M+1/(νPEPCK-M+1 + νGlyc+1). The 13C enrichment of the product of an enzymatic reaction is determined by the 13C enrichment of the precursor pool. Because the 13C enrichment of PEP (PEPAPE) and its precursor pool (malateAPE) can be measured, then the ΦPEPCK-M is determined simply by 100 × PEPAPE/MalateAPE as long as there is a 1:1 relationship between the product and precursor labeling. All data are reported as the mean ± S.E. Unpaired two-tailed Student t tests and one-way ANOVA were performed using the prism software package version 5 (GraphPad). Differences were considered to be significant at p < 0.05. Previous reports have confirmed the absence of any significant PEPCK-C activity in islets, but little is known about PEPCK-M (12MacDonald M.J. McKenzie D.I. Walker T.M. Kaysen J.H. Horm. Metab. Res. 1992; 24: 158-160Crossref PubMed Scopus (68) Google Scholar, 13MacDonald M.J. Chang C.M. Diabetes. 1985; 34: 246-250Crossref PubMed Scopus (36) Google Scholar). PEPCK-M mRNA was 15-fold higher relative to PEPCK-C in INS-1 832/13 cells (Fig. 2A) and 11-fold higher level in rat islets (Fig. 2B). The relative abundance of PEPCK-M message was 97.1 ± 0.3% of SCS-GTP and 20 ± 0.4% of β-actin in INS-1 832/13 cells. Western blotting demonstrated no significant PEPCK-C in INS-1 832/13 cells, mouse, or rat islets and confirmed its presence in primary rat hepatocytes and whole rat liver (Fig. 2E). In contrast, robust PEPCK-M expression was detected in INS-1 832/13 cells, rat, and mouse islets at a level comparable with that observed in the hepatocytes and liver. There is conflicting evidence among PEPCK enzymatic activity determinations for the significant presence versus the absence of PEPCK in rodent islets (12MacDonald M.J. McKenzie D.I. Walker T.M. Kaysen J.H. Horm. Metab. Res. 1992; 24: 158-160Crossref PubMed Scopus (68) Google Scholar, 13MacDonald M.J. Chang C.M. Diabetes. 1985; 34: 246-250Crossref PubMed Scopus (36) Google Scholar, 14Hedeskov C.J. Capito K. Thams P. Biochim. Biophys. Acta. 1984; 791: 37-44Crossref PubMed Scopus (7) Google Scholar, 15Hedeskov C.J. Capito K. Horm. Metab. Res. Suppl. 1980; 10: 8-13PubMed Google Scholar). Where no PEPCK activity was detected, the assay was a single end point measurement of the forward reaction in the direction of PEP formation using Mg-ITP as reactant in the absence of any inhibitors (13MacDonald M.J. Chang C.M. Diabetes. 1985; 34: 246-250Crossref PubMed Scopus (36) Google Scholar). A particular concern of the forward reaction is that the product can be rapidly depleted by either contaminating enolase or PK activity leading to gross underestimations of enzyme activity. This is especially important because the IDP formed by PEPCK can lead to consumption of PEP by PK whose activity is extremely high in insulin-secreting cells (13MacDonald M.J. Chang C.M. Diabetes. 1985; 34: 246-250Crossref PubMed Scopus (36) Google Scholar, 16Bârzu O. Abrudan I. Proinov I. Kiss L. Ty N.G. Jebeleanu G. Goia I. Kezdi M. Mantsch H.H. Biochim. Biophys. Acta. 1976; 452: 406-412Crossref PubMed Scopus (20) Google Scholar, 17Hohnadel D.C. Cooper C. FEBS Lett. 1973; 30: 18-20Crossref PubMed Scopus (14) Google Scholar, 18Plowman K.M. Krall A.R. Biochemistry. 1965; 4: 2809-2814Crossref PubMed Scopus (66) Google Scholar). For all our measurements, the reverse reaction (in the direction of OAA formation) was used whereby OAA production is coupled to malate dehydrogenase where NADH oxidation to NAD+ is observed in real time. Specificity of these reactions is conferred by comparison of the activities in the presence and absence of concentrated CO2. dGDP was used because of its preferential use by PEPCK versus PK. Inhibitors were used to avoid nonspecific NADH oxidation by Complex 1 (rotenone) and lactate dehydrogenase (by blocking pyruvate production via inhibition of PK with phenylalanine (Phe)) (6Wiese T.J. Lambeth D.O. Ray P.D. Comp. Biochem. Physiol. B. 1991; 100: 297-302Crossref PubMed Scopus (23) Google Scholar, 7Petrescu I. Bojan O. Saied M. Bârzu O. Schmidt F. Kühnle H.F. Anal. Biochem. 1979; 96: 279-281Crossref PubMed Scopus (102) Google Scholar). The enolase inhibitor NaF was used to prevent loss of PEP because of enolase activity. Using this strategy, substantial PEPCK activity was measured in INS-1 832/13 (51 ± 8%) cells and rat islets (76 ± 1%) compared with primary rat hepatocytes (Fig. 2D). In contrast to whole cell activities, mitochondria PEPCK activities were higher in insulin-secreting cells than hepatocytes; that is, INS-1 832/13 cells having PEPCK activity 470 ± 20% of mitochondria from hepatocytes, although islets were 550 ± 30% higher. PEPCK-M and PEPCK-C have many similarities in enzyme activities but display notable differences in νmax depending on the pH and the counter ion(s) utilized as well as the Km of CO2 as might be expected for their unique subcellular environments (19Holyoak T. Nowak T. Biochemistry. 2004; 43: 7054-7065Crossref PubMed Scopus (8) Google Scholar, 20Ballard F.J. Hanson R.W. Reshef L. Biochem. J. 1970; 119: 735-742Crossref PubMed Scopus (47) Google Scholar, 21Goto Y. Shimizu J. Okazaki T. Shukuya R. J. Biochem. 1979; 86: 71-78PubMed Google Scholar, 22Holten D.D. Nordlie R.C. Biochemistry. 1965; 4: 723-731Crossref PubMed Scopus (58) Google Scholar, 23Ishihara N. Kikuchi G. Biochim. Biophys. Acta. 1968; 153: 733-748Crossref PubMed Scopus (19) Google Scholar, 24Sato A. Suzuki T. Kochi H. J. Biochem. 1986; 100: 671-678Crossref PubMed Scopus (10) Google Scholar). Of note, Mg2+ reduces PEPCK-M activity, whereas Mn2+ is the preferred divalent. Mn2+ was used for comparisons of subcellular localization of PEPCK to prevent underestimation of PEPCK-M activity. Direct measurements of PEPCK activity were made in the cytosolic and mitochondrial fractions with glutamate dehydrogenase as a marker for contamination of mitochondrial matrix components. Virtually all of the PEPCK activity from cells and islets was localized to the mitochondrial fraction when adjusted for leakage of mitochondrial contents into the cytosol fraction by parallel measurements of glutamate dehydrogenase activity from the same samples (Fig. 2E). Simply the presence of enzyme activity does not necessarily connote an important physiologic role; therefore, the intracellular function of PEPCK-M was queried. Because PEPCK-M is compartmentalized in the mitochondrial matrix where the concentrations of substrate and products may be locally high and different from the cytosol, direct comparison of these activities ex vivo in lysates may be misleading. Thus, a labeling strategy was developed to measure PEPCK-M flux in intact cells to assess the functional contribution of PEPCK-M to metabolism. The two primary sources of PEP synthesis are the glycolytic enolase reaction of 2-PG and the cataplerotic PEPCK reaction (Figs. 1 and 3A). Because insulin-secreting cells lack PEPCK-C (Fig. 2 and Refs. 12MacDonald M.J. McKenzie D.I. Walker T.M. Kaysen J.H. Horm. Metab. Res. 1992; 24: 158-160Crossref PubMed Scopus (68) Google Scholar and 13MacDonald M.J. Chang C.M. Diabetes. 1985; 34: 246-250Crossref PubMed Scopus (36) Google Scholar)), any PEPCK activity measured in β-cells will arise from PEPCK-M. INS-1 832/13 cells, unlike islets, have high monocarboxylate transporter activity, so 13C-labeled pyruvate can be used to assess PEPCK-M flux in living cells. After entry into the mitochondria, pyruvate carboxylase (PC) can metabolize [3-13C]pyruvate into OAA that becomes racemized across carbons 2 and 3 as a consequence of rapid equilibration across fumarase (Fig. 3A). Pyruvate dehydrogenase metabolism of [3-13C]pyruvate with continued TCA cycle flux will lead to a similar labeling pattern of OAA. Any labeled pyruvate that spontaneously decarboxylated to form [2-13C]acetate would similarly label OAA if incorporated into [2-13C]acetyl-CoA. Therefore, regardless of the direction of mitochondrial metabolism (PC versus pyruvate dehydrogenase), the OAA pool will be enriched equally in the 2 and 3 position during the first cycle. Because OAA and malate are in isotopologue equilibrium with each other, the 13C enrichment of the more abundant and less labile malate reflects the enrichment of OAA. Thus, [3-13C]pyruvate can specifically mass-label the PEPCK precursor pool as determined by measuring the M+1 APE of malate by LC/MS/MS. As the OAA is metabolized to PEP by PEPCK, 100% of the 13C label will be transferred to the respective 2 or 3 position with the loss of unlabeled CO2. The newly synthesized PEP will have the same mass isotopologue enrichment as its precursor OAA. Thus, by knowing the precursor pool enrichment of OAA (as measured by the APE of its surrogate, malate) it is possible to calculate the fractional contribution of PEPCK-M to the cellular PEP pool (ΦPEPCK-M). INS-1 832/13 cells were incubated in 5 mm [3-13C]pyruvate with G0, G3, G7, or G15 mm unlabeled glucose. As the glucose concentration increases, both glycolytic and mitochondrial metabolite pools expand. As expected the concentration (combined labeled and unlabeled) of PEP and malate increased with increasing concentrations of glucose. Compared with 0 mm glucose, malate concentrations doubled, whereas PEP increased more than 20-fold at 15 mm glucose (Fig. 3B). Similarly, the -fold increase from G0 in 2-PG/3-PG that is in exchange with PEP was 2.0 ± 0.3-fold at G3, 3.9 ± 0.4-fold at G7, and 14.4 ± 1.0-fold at G15. Unlike malate, the aspartate pool that is not in direct chemical exchange with malate shrunk as expected to 0.76 ± 0.02 at G3, 0.47 ± 0.02-fold at G7, and to 0.26 ± 0.2-fold at G15 compared with G0. In contrast to the large changes in concentrations noted above, in the absence of 13C label, the enrichment of the M+1 isotopologues of malate, aspartate, PEP, and 2-PG/3-PG approximated natural abundance predictions (supplemental Fig. S1) with the exception of PEP at G0, where the base-line contribution to M+1 was higher because of the low signal-to-noise ratio. In the presence of 13C-labeled pyruvate, the enrichment of malate was 55%, with a small but significant decrease in enrichment as the glucose concentration was increased to 15 mm, presumably as