Magnetic resonance spectroscopic investigation of mitochondrial fuel metabolism and energetics in cultured human fibroblasts: Effects of pyruvate dehydrogenase complex deficiency and dichloroacetate

Molecular Genetics and Metabolism 89 (2006) 97–105 www.elsevier.com/locate/ymgme

Magnetic resonance spectroscopic investigation of mitochondrial fuel metabolism and energetics in cultured human Wbroblasts: EVects of pyruvate dehydrogenase complex deWciency and dichloroacetate Nicholas E. Simpson a,¤, Zongchao Han a, Kristen M. Berendzen a, Carol A. Sweeney a, Jose A. Oca-Cossio a, Ioannis Constantinidis a, Peter W. Stacpoole a,b,c a

Department of Medicine, Division of Endocrinology and Metabolism, University of Florida, Gainesville, FL 32610, USA b Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA c General Clinical Research Center, University of Florida, Gainesville, FL 32610, USA Received 25 April 2006; accepted 26 April 2006 Available online 12 June 2006

Abstract The pyruvate dehydrogenase complex (PDC) is integral to metabolism and energetics. Congenital PDC deWciency leads to lactic acidosis, neurological degeneration and early death. An investigational compound for such defects is dichloroacetate (DCA), which activates the PDC (inhibiting reversible phosphorylation of the E1 subunit) and decreases its turnover. Here, primary human Wbroblast cultures from Wve healthy subjects and six patients with mutations in the PDC-E1 component were grown in media § DCA, exposed to media containing 13C-labeled glucose, and studied (as cell extracts) by nuclear magnetic resonance (NMR) spectroscopy. Computer modeling of NMR-derived 13C-glutamate isotopomeric patterns estimated relative carbon Xow through TCA cycle-associated pathways and characterized eVects of PDC deWciency on metabolism and energetics. Rates of glucose consumption (GCR) and lactate production (LPR) were measured. With the exception of one patient cell line expressing an unusual splicing mutation, PDC-deWcient cells had signiWcantly higher GCR, LPR and label-derived acetyl-CoA, indicative of increased glycolysis vs. controls. In all cells, DCA caused a major shift (40% decrease) from anaplerotic-related pathways (e.g., pyruvate carboxylase) toward Xux through PDC. Ignoring the patient with the splicing mutation, DCA decreased average glycolysis (29%) in patient cells, but had no signiWcant eVect on control cells, and did not change LPR or the nucleoside triphosphate to diphosphate ratio (NTP/NDP) in either cell type. Maintenance of NTP despite reduced glycolysis indicates that DCA improves metabolic eYciency by increasing glucose oxidation. This study demonstrates that NMR spectroscopy provides insight into biochemical consequences of PDC deWciency and the mechanism of putative therapeutic agents. © 2006 Elsevier Inc. All rights reserved. Keywords: NMR spectroscopy; Fibroblasts; Mitochondria; Stable isotopes; Pyruvate dehydrogenase complex deWciency; Dichloroacetate

Introduction Mitochondrial diseases are among the most challenging inborn errors of metabolism to evaluate biochemically. Contributing factors include the heteroplasmic distribution of mitochondrial DNA mutations, tissue-speciWc expression of nuclear gene defects, variability in in vitro diagnostic enzymologic techniques and over-reliance on readily *

Corresponding author. Fax: +1 352 846 2635. E-mail address: [email protected] (N.E. Simpson).

1096-7192/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2006.04.015

measurable, but relatively nonspeciWc, in vivo surrogate disease markers, such as the blood lactate concentration [1]. Furthermore, once a biochemical or molecular genetic diagnosis is made, there is usually little subsequent investigation of the intracellular metabolic consequences of the enzyme deWciency. For example, although defects in the pyruvate dehydrogenase complex (PDC) are among the most common biochemically proven causes of congenital lactic acidosis [2], there have been few quantitative studies of the eVects of PDC deWciency on mitochondrial fuel metabolism and energetics.

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Primary cultures of skin Wbroblasts are frequently employed to diagnose PDC deWciency [3,4], as well as primary disorders of fatty acid oxidation [5] and, under certain conditions, of electron transport [6–9]. Fibroblasts may also serve as a readily available tissue to investigate the biochemical phenotype of speciWc mitochondrial diseases. In this regard, nuclear magnetic resonance (NMR) spectroscopy has been shown to be an excellent ‘toolbox’ for investigation of inborn errors of metabolism [10] and oVers a non-invasive, sensitive and quantitative means for accomplishing this objective. 13C NMR spectroscopy may be utilized to investigate the metabolic fate of substrates isotopically enriched with 13C. Because the metabolic paths by which 13C-labeled carbons enter (and re-enter) the TCA cycle dictate the resultant label positions, contributions of metabolic pathways and indices associated with the tricarboxylic acid (TCA) cycle can be determined by analyzing the isotopomeric patterns of the carbons of glutamate, a TCA cycle byproduct formed from -ketoglutarate (-KG) [11–13]. Such complex spectral (isotopomer) patterns arise when adjacent carbons are labeled with 13C, due to spin–spin interactions. Likewise, 31P NMR spectroscopy is commonly employed to investigate cellular bioenergetics. Over the past few years, NMR spectroscopy has been utilized to probe structure–function relationships in respiratory chain complexes [14], the eVects of trauma [15], stress [16], toxins [17], and chemotherapeutic agents [18,19] on mitochondrial function in vitro, and the consequences of aging [20], insulin resistance [21], and mitochondrial DNA mutations or enzyme deWciencies [22–26] on oxidative phosphorylation in vivo. In this study, we used 13C and 31P NMR spectroscopic methods to investigate the consequences of PDC deWciency on glucose metabolism and bioenergetics, using cultured skin Wbroblasts from healthy subjects and patients with

deWned mutations in the E1 component of the PDC. We also determined the intramitochondrial eVects of dichloroacetate (DCA), an activator of the PDC, in normal and defective cells. Materials and methods These studies were approved by the Institutional Review Board, University of Florida.

Cell lines Human Wbroblast cultures were obtained by skin biopsy from Wve healthy subjects and six patients with proven defects in the E1 or E1 subunit of PDC (Table 1). Cells were cultured as monolayers in Dulbecco’s modiWed Eagle’s medium, DMEM (Mediatech, Herndon, VA), containing 20 mM D-glucose and was supplemented with 20% (v/v) fetal bovine serum (Hyclone, Logan, UT), antibiotics (100 U/ml penicillin and 100 g/ml streptomycin), insulin-transferrin (5 g/ml)-sodium selenite (5 ng/ml) solution (Sigma, St. Louis, MO), and 6 mM L-glutamine (Sigma, St. Louis, MO). Cells were cultured under the media conditions described above (untreated), and then exposed to media containing 5 mM dichloroacetate (DCA; Sigma, St. Louis, MO) for 1 day, or cultured in the presence of 5 mM DCA for 30 days (at least three passages). Cultures were grown at 37 °C under humidiWed (5% CO2/95% air) conditions. 13

C labeled media

ConXuent Xasks of Wbroblasts were exposed to complete DMEM containing 15 mM uniformly labeled 13C-glucose (Cambridge Isotopes, Cambridge, MA) with or without 5 mM DCA and were incubated for 24 h to assure that a steady-state 13C signal was reached in these slowly metabolizing cells.

Extracts ConXuent T-175 Xasks (7–10) were used for each extraction. A representative Xask of each experiment was used to estimate cell number and viability by manual cell counting and trypan blue exclusion. After incubation,

Table 1 Biochemical characteristics of cell lines Cell type

Subject

Gender

PDC activitya

Control

A B C D E

M M M F M

2.25 § 0.44 2.42 § 0.04 2.17 § 0.29 2.53 § 0.15 2.46 § 0.14

Mean § SD

2.37 § 0.15

F F F F F M

0.91 § 0.16 1.28 § 0.40 0.90 § 0.17 1.05 § 0.34 1.11 § 0.39 1.49 § 0.08

Mean § SD

1.12 § 0.23¤

Patient

1 2 3 4 5 6

Subunit

Nucleotide change

Amino acid change

Functional class

E1 E1 E1 E1 E1 E1

c.642G > T exon 7 c.642G > T exon 7 c.1063–1068 exon 11 c.904C > T exon 10 c.438A > G exon 6 g.592G > A exon 6

p.Trp214Cys p.Trp214Cys p.Arg355 p.Leu356 p.Arg302Cys p.Glyl46Gly N/A

Missense Missense Deletion Missense Substitutionb Mis-splicingc

Skin biopsy cells from healthy volunteers and patients with PDC deWciency were cultured as described in Methods. Data are means § SD of quadruplicate determinations. Mutations are expressed using the Human genome variation society (HGVS) nomenclature. a PDC activity is expressed in (nmol) 14CO2/min/mg protein. b The resultant protein is less stable, probably due to enhanced ubiquitin-mediated protein degradation (unpublished observation). c This alteration is thought to create an SRp40-speciWc exonic splicing enhancer site. ¤ p ¿ 0.001.

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dual-phase extractions were performed [27] as follows. Flasks were rinsed with ice-cold saline to remove residual extracellular media, extracted with ice-cold methanol and scraped oV the Xask. The methanol cell slurry was collected and equivalent volumes of chloroform and water were added. The aqueous portion of each extract was isolated, treated with Chelex-100 (Sigma, St. Louis, MO), lyophilized, resuspended in 450 l of D2O (Cambridge Isotopes, Cambridge, MA), and placed in a 5 mm NMR tube.

Analytical techniques Media samples were withdrawn from each Xask at the start and conclusion of the exposure period and assayed for glucose and lactate concentrations, using a VITROS DT60 II Bioanalyzer (Ortho-Clinical Diagnostics, Rochester, NY). Rates of glucose consumption (GCR) and lactate production (LPR) were calculated from the change in the amount of glucose and lactate (amount D volume ¡ concentration), respectively, during the 24 h incubation. These rates were normalized to a unit of 105 cells present within the Xasks.

NMR Spectroscopy 1

H-decoupled 13C and 31P NMR spectra were acquired using a 5 mm broadband receiving coil in an 11.75 Tesla vertical bore Bruker Avance500 (Bruker, Billerica, MA). An insert containing dioxane (13C) and trimethylphosphate (31P) acted as a chemical shift reference. The acquisition parameters were: 13C-sweep width D 30 kHz; repetition time D 6 s; number of transients D 10240; 31P-sweep width D 8.09 kHz; repetition time D 6 s; number of transients D 1024. Waltz 1H decoupling was applied throughout the 13C and 31P acquisitions.

Spectroscopic analysis Areas under the 31P resonances of the -NTP and -NDP were determined by line-Wt analysis with the program ‘Nuts’ (Acorn NMR, Fremont, CA) and NTP/NDP ratios were calculated. 13C NMR spectra were analyzed by calculating the multiplet areas of the C2, C3, and C4 glutamate resonances by line-Wt analysis (Acorn NMR, Fremont, CA). Correction factors were applied to glutamate signal areas to correct for relaxation and nuclear Overhauser eVects. Isotopomeric modeling analysis using the multiplet patterns and the C3/C4 glutamate ratio was performed with tcaCALC [11–13], as described below.

Metabolic model The modeling program tcaCALC uses algebraic equations to describe glutamate isotopomeric patterns in terms of pathway Xuxes of an applied metabolic model. The model we applied (Fig. 1) includes two anaplerotic entrances to the TCA cycle: the anaplerotic entrance of pyruvate to oxaloacetate (OAA) via pyruvate carboxylase (PC), and a non-PC anaplerotic entrance from undeWned labeled carbon sources. Other enzymes included in the model are the PDC, and enzymes that convert carbons from the TCA cycle to pyruvate, here termed pyruvate kinase (PK), that also includes contributions from phosphoenolpyruvate carboxykinase (PEPCK) and malic enzyme (ME). Monte-Carlo simulations and non-linear least-squares analysis of the isotopomeric patterns provided the ratio of pyruvate metabolized by PC vs. PDC; the fraction of labeled pyruvate carbons; the % of acetyl-CoA derived from pyruvate; the replenishment of pyruvate from TCA cycle intermediates; and the amount of non-PC anaplerosis contributing to the TCA cycle. The % anaplerosis is deWned as the relative carbon entry from both anaplerotic pathways to the total carbon entering the TCA cycle. Pyruvate cycling is deWned as the average of the rates of anaplerotic entrance into the TCA cycle and the exit from the TCA cycle to the pyruvate pool [28,29].

PDC activity PDH complex activity was measured by the rate of 14C formation from [1- C]-labeled pyruvate [30] as follows. Fibroblasts harvested by trypsin14

Fig. 1. The metabolic model used in analyses. The parameters PDC, PC, and PK are tcaCALC parameters representing the relative pyruvate metabolism (PDH and PC) and synthesis from TCA cycle intermediates (PK). Abbreviations used in this Wgure include: PDC, pyruvate dehydrogenase complex; PC, pyruvate carboxylase; PK, pyruvate kinase; OAA, oxaloacetate; -KG, -ketoglutarate; TCA, tricarboxylic acid. ization from a conXuent T-175 Xask were washed in phosphate buVered saline (PBS), then resuspended in a buVer (PBS) containing serine protease inhibitors (Leupeptin and PMSF). Cells were incubated for 15 min at 37 °C with DCA (Wnal concentration of 3 mM) to activate the PDC, then reactions were halted by addition of a stop solution (25 mM NaF, 25 mM EDTA, 4 mM dithiothreitol, 40% ethanol), and cells lysed by repeated freezing and thawing. Sixteen aliquots of each Wbroblast sample were assayed in test tubes for PDC activity; four blanks (lysate and water), and four tests (lysate and water containing thiamine pyrophosphatate and Coenzyme A) were assayed at reaction times of 5 and 10 min. A 14C-labeled pyruvate aliquot of known radioactivity was added to the cell lysates and the test tubes were immediately closed with a rubber stopper outWtted with a center well (Kimble-Kontes, Vineland, NJ) containing 1 cm2 chromatography paper soaked with 100 l hyamine hydroxide. Reactions were halted by adding stopping buVer. The 14CO2 evolved due to PDC-mediated catalysis and trapped in the paper was determined by liquid scintillation. SpeciWc PDC activity was expressed as nmol 14CO2 produced/min/mg protein. Excess cell lysate was used to determine total protein.

Mutational analyses Fibroblast mRNA was extracted (RiboPure™ kit; Ambion, Austin, TX) and cDNA was made with a Qiagen Omniscript RT kit according to manufacturer’s instructions (Qiagen, Valencia, CA). The speciWc target (GenBank Accession No. NM_000284) was ampliWed with a pair of primers that bracket the entire coding portion of E1. cDNA was ampliWed by polymerase chain reaction using Pfx DNA polymerase (Invitrogen, Carlsbad, CA) with forward primer (bases 22–41) PDHA-L (5⬘-gggcacctgaaggagacttg-3⬘) and reverse primer (bases 1443–1469) PDHA-R (5⬘-cactc aataattcatcttttaatgcac-3⬘). Products were assessed by running on a 1% agarose gel, puriWed with a Qiagen QIAquick puriWcation kit and sequenced (Perkin-Elmer/Applied Biosystems, Wellesley, MA) by the DNA Sequencing Core at the University of Florida.

Statistical methods Comparison of NMR-derived bioenergetic variables (e.g., % label in pyruvate pool; % acetyl-CoA from pyruvate; PC/PDC; NTP/NDP) were determined between untreated control and patient cell populations by Student’s two-tailed paired t-test. The signiWcance of change in the above-

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described bioenergetic variables associated with DCA exposure was tested with the parametric group-paired t-test.

Results Table 1 summarizes the biochemical characteristics of the cell lines. The mean residual enzyme-speciWc activity in patient cells was 47% of that determined in cells from healthy subjects, and ranged from 38% to 63% of normal. Four subjects (patients 1–4) had a missense or deletion mutation in the E1 subunit of the PDC. Another (patient 5) had a novel defect in the  subunit of E1, perhaps contributing to its instability due to an enhanced ubiquitinmediated degradation of this protein, the details of which will be reported separately (Han et al., unpublished observations). Patient 6 expressed somatic mosaicism for a missense mutation in the coding region for the E1, causing mis-splicing and non-expression of the E1 protein subunit [31]. Clinical characteristics of the patients at the times of diagnosis are outlined in Table 2. All patients presented early in life with developmental delay. Microcephaly was a common feature. At the time of diagnosis, lactate levels were elevated in the cerebrospinal Xuid (range: 3.5– 7.1 mmol/l) and blood (range: 1.3–7.4 mmol/l). Fig. 2 displays representative 31P spectra (normalized to the reference intensity and to approximate cell numbers) from control and PDC-deWcient cell lines. High and low energy phosphorus-containing metabolites observed include nucleoside di- and tri-phosphates (NDP and NTP), phosphocreatine (PCr), inorganic phosphate (Pi), and the membrane-related phosphomonoesters (PMEs; phosphocholine and phosphoethanol amine) and phosphodiesters (PDEs; glycerophosphocholine and glycerophosphoethanol amine). Normalized spectra in Fig. 2 from untreated normal (panel a) and PDC-deWcient (panel b) cultures showed similar levels of NTP and NDP. The duration of DCA exposure had no eVect on the NTP/NDP ratio, and results following 30-day DCA exposures are reported here. Spectra from control and patient cultures exposed to DCA are shown in panels c and d. There were no diVerences in NTP or NDP. However, cell lines treated with DCA showed an increase in both PMEs and PDEs. The signal-tonoise diVerences among the spectra shown in Fig. 2 were predominantly due to the number of cells present in each sample, as PDC-deWcient cells were more diYcult to expand in culture than were control cells. Provided there is ade-

Fig. 2. 31P NMR spectra comparing a control cell line and a PDC-deWcient cell line exposed to complete media containing 15 mM uniformly labeled 13 C-glucose without (a and b) and with (c and d) 5 mM DCA. Assignments are placed above the resonances, and include: sugar phosphates (“SP”); the phosphomonoesters (PME), phosphoethanolamine (PE), and phosphocholine (PC); inorganic phosphate (Pi); an unknown decomposition from the reference tube (“x”); the phosphodiesters (PDE), glycerophosphoethanolamine (GPE), and glycerophosphocholine (GPC); phosphocreatine (PCr); the nucleoside triphosphates (-, - or -NTP); the nucleoside diphosphates (- or -NDP); nicotinamide adenine dinucleotide (NAD). Strikeouts indicate cropped resonances.

quate signal-to-noise, the NTP/NDP values are independent of cell number. Visual inspection of 13C spectra revealed diVerences between control cells and PDC-deWcient cells in the integration of the carbon label, as shown in the representative spectra in Fig. 3 (panels a and b). Glutamate carbons were well labeled in both control and deWcient cells, thereby permitting analysis of metabolic processes related to the TCA cycle. However, other amino acids, such as serine and alanine, were typically not well labeled in the patient cells, indicating that either little energy was expended toward the synthesis of these amino acids, or that they may be metabolized as fuel

Table 2 Clinical characteristics of patients Subject

Gender

Age at diagnosis

Initial clinical features

CSF lactate (mmol/1)

Blood lactate (mmol/1)

1 2 3 4

F F F F

l year 7 months l year 7 months l year 10 months l year 5 months

Unknown 3.5 7.1 4.2

5 1.3 2.1 5.8

5 6

F M

4 years 0 month 2 years 6 months

Developmental delay; failure to thrive; microcephaly; cortical atrophy Developmental delay; failure to thrive Developmental delay; microcephaly; agenesis of the corpus callosum Developmental delay; microcephaly; hypotonia; ataxia Developmental delay; microcephaly; tachypnea; agenesis of the corpus callosum Developmental delay; microcephaly; mild hypotonia; spasticity

6.9 3.5

7.4 2.9

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Fig. 3. Proton-decoupled 13C NMR spectra comparing a control cell line and a PDC-deWcient cell line exposed to complete media containing 15 mM uniformly labeled 13C-glucose without (a and b) and with (c and d) 5 mM DCA. Assignments are placed above the resonances and include the carbon labeled. Strikeouts indicate cropped resonances.

sources. Again, the signal-to-noise diVerences between the acquired spectra were in part due to the number of cells in the samples, but as suYcient signal was present, they have no bearing on the results of the isotopomeric analysis. DCA altered intermediary metabolism and glutamate isotopomeric patterns in both control (panel c) and patient (panel d) cells. However, the duration of DCA exposure had no eVect on how carbons entered the TCA cycle, and the results derived from cells following 30-day DCA exposures are shown. 13C spectra from control cells exhibited enhanced carbon integration into glutamate, and all cell lines showed a reduction in lactate levels. Fig. 4 demonstrates the representative isotopomeric changes to glutamate carbons (C2, C3, and C4) that are visible as a consequence of exposure to DCA (here, from extracts of control line “C” and patient line “2”). In the absence of DCA, the singlets were more prominent (indicating less contiguous carbon labeling). Exposure to DCA resulted in more quartets and triplets (where applicable) being present in the spectra. Isotopomeric changes to the patient cell line as a result of DCA exposure were less striking than the changes observed in the control cell lines. These isotopomeric patterns hold key information regarding entrance of the labeled substrate into the TCA cycle, and were analyzed with tcaCALC. Table 3 compares indices of carbon and energy metabolism derived from untreated control cells and PDC-deWcient cells. The mean GCR and LPR values were higher in PDC-deWcient cells, suggesting that the rate of anaerobic

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Fig. 4. Isotopomer patterns of glutamate resonances obtained from extracts of control and PDC-deWcient Wbroblast cultures exposed to complete media containing 15 mM uniformly labeled 13C-glucose §5 mM DCA. Letters under the spectra denote the origin of the peaks: S represents the singlet; D, the doublet; and Q, the quartet.

Table 3 Baseline biochemical measures and tcaCALC-derived indices in normal and PDC-deWcient cell lines Indices

Cell Lines Control (n D 5)

GCR nmol/h/105 cells LPR nmol/h/105 cells NTP/NDP Fraction of acetyl-Co A from pyruvate Carbon to TCA cycle through PC vs. PDCa Carbon from TCA cycle to pyruvatea Carbon to TCA cycle via non-PC anaplerosisa Pyruvate cyclinga Carbon to TCA cycle via total anaplerosisa % Carbon entrance to TCA cycle via anaplerosis

PDC-deWcient (n D 6)

73.3 § 45.5 97.8 § 49.9 7.0 § 2.6 92.7 § 7.2% 1.28 § 1.24

100.3 § 60.2 141.1 § 81.2 7.5 § 2.2 92.2 § 14.0% 0.61 § 0.43

1.42 § 1.23 1.39 § 0.36

1.07 § 0.46 1.38 § 0.65

1.35 § 1.23 2.66 § 1.44

0.84 § 0.43 1.99 § 1.00

69.4 § 9.8%

62.2 § 15.5%

a Values of indices are relative to the carbon entrance to the TCA cycle from acetyl-CoA (via citrate synthase), being deWned arbitrarily as 1. Abbreviations are: GCR, glucose consumption rate; LPR, lactate production rate; PC, pyruvate carboxylase; PDC, pyruvate dehydrogenase complex; TCA, tricarboxylic acid; NTP, nucleoside triphosphate; NDP, nucleoside diphosphate; CoA, coenzyme A. Each cell line was run in duplicate, therefore data are expressed as means § SD of 10 or 12 determinations, for control or deWcient lines, respectively.

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metabolism increased compared to control cells. However, these diVerences were not statistically signiWcant due to the contribution from the cells with the mis-splicing mutation (patient 6). The GCR and LPR of patient 6 cells were 13.8 and 27.0 nmol/h/105 cells, respectively, far lower than even the control cell values. Excluding the mis-splicing mutation yields PDC-deWcient averages of 125.0 § 40.4 and 173.6 § 56.4 for the GCR and LPR, respectively, both signiWcantly higher than the control values (p < 0.05 and p < 0.02, respectivity). Other striking anomalies associated with the mis-splicing mutation were a reduced fraction of acetyl-CoA from pyruvate (70% compared to 93% and 99% for the average control and remaining PDC-deWcient cells, respectively), a greatly reduced fraction of carbon entering the TCA cycle through PC vs. PDC (0.13 compared to 1.28 and 0.75 for control and other PDC-deWcient cells, respectively), and a drop in carbon going from the TCA cycle to

pyruvate (0.54 compared to 1.42 and 1.23 for control and PDC-deWcient cells, respectively). However, the ratio of NTP/NDP did not diVer between controls or PDC-deWcient cells with or without inclusion of patient 6. Of the tcaCALC-derived measures of metabolic activity, only the fraction of acetyl-CoA derived from pyruvate diVered signiWcantly (p < 0.05) between cell types (when the mis-spliced mutation is excluded), with nearly all acetyl-CoA from the PDC-deWcient cell lines arising from the labeled pyruvate. This indicates that the pyruvate pool leading to acetyl-CoA in untreated PDC-deWcient cells is completely labeled, probably as a consequence of enhanced glycolysis, but also potentially due to reduced synthesis of acetyl-CoA from other substrates (e.g., acyl groups). Fig. 5 summarizes changes in measured and tcaCALCderived indices of metabolism due to 30-day exposure to DCA. If patient 6 is excluded, there was a trend toward a

Fig. 5. Depiction and summary of changes in measured and tcaCALC-derived indices of metabolism due to 30-day exposure to 5 mM DCA. The average % change is indicated, and signiWcant changes between unexposed and DCA-exposed cultures (as determined by group-paired t-tests) are denoted in parentheses. N.S. denotes not signiWcant.

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reduction in GCR (¡29.1 § 31.9%; p D 0.10) in PDC-deWcient cells, suggesting that DCA inhibited glycolysis; inclusion results in a non-statistical 9.6% reduction. Moreover, neither the LPR nor the NTP/NDP ratio was signiWcantly aVected by DCA in either normal or deWcient cell lines. The baseline values and DCA-induced changes in the metabolic indices obtained through analysis of the glutamate isotopomers were consistent among PDC-deWcient cells except for patient 6, and in the absence of this patient demonstrated a mean 8.5 § 4.7% decrease (p < 0.02) in the fraction of acetylCoA derived from the pyruvate pool following DCA exposure. This indicates DCA may increase the conversion of alternate substrates (e.g., acyl groups) to acetyl-CoA, decrease the rate of glycolysis, or exert both eVects simultaneously. The most marked eVect of DCA was on pathways associated with anaplerosis through PC. All cell lines exposed to DCA except for patient 6 demonstrated a reduction (range, ¡8 to ¡74%; average, ¡39.3 § 24.7%) in the Xow of carbons to the TCA cycle from the anaplerotic enzyme PC relative to PDC (p < 0.001). Patient 6 showed the opposite (60% increase in Xow to PC), resulting in a mean decrease for all cells of 30.3 § 37.9% (p < 0.05). This is consistent with the known stimulatory eVect of DCA on PDC activity. Mean total anaplerosis also was reduced 17.4 § 19.6% (p < 0.02) in DCA treated cells, due to the relative decrease in Xow through PC. The Xow of carbons to the pyruvate pool from the TCA cycle decreased in all cells except for a 28.9% increase in those from patient 6, following exposure to DCA (range, ¡22.8 to ¡52.4%; average, ¡37.4 § 14.0%, p < 0.001). This decrease in carbon Xow, coupled to the reduction in Xow through PC, led to a reduction in pyruvate cycling (range, ¡21.5 to ¡59.9; average, ¡38.2 § 13.2%, p < 0.0001) in all cell lines (except patient 6) treated with DCA. If the pyruvate cycling value for patient 6 is included (an increase of 35.2%), the overall reduction is still maintained (average, ¡31.6 § 25.5%, p < 0.005). Discussion These data indicate that the application of NMR spectroscopy to primary cultures of skin Wbroblasts may be useful in delineating the biochemical consequences of PDC deWciency and the action of putative therapeutic agents thereon. Although our interpretation is limited primarily by the few number of cell lines examined, the following conclusions appear warranted. First, robust 13C and 31P NMR spectra, indicative of oxidative metabolism and bioenergetics, can be generated in cultured skin Wbroblasts. The use of uniformly labeled 13C-substrate and isotopomeric modeling analysis provides insight into the fate of glucose carbon in healthy and PDC-deWcient cells. In this regard, we found that patient cells demonstrated increased rates of glucose consumption and lactate production, consistent with a greater than normal reliance on glycolysis for NTP production. In fact, under basal conditions, the similar NTP/ NDP ratio in normal and PDC-deWcient cells suggests

103

that glycolysis in deWcient cells is capable of maintaining adequate energy stores. The PDC-deWcient cells studied had suYcient residual enzyme activity to allow substantial glucose-derived pyruvate to be decarboxylated, thereby providing the TCA cycle with acetyl-CoA, mainly from glucose. Whether more severely enzyme deWcient cells would require alternative sources of acetyl-CoA for adequate TCA cycle activity remains to be investigated. Moreover, the small sample size of PDC-deWcient cells precludes insight into possible diVerential eVects of speciWc E1 mutations or mutations of other genes that encode the complex on the indices of glucose and energy metabolism described here. The unusual glucose consumption and NMR-derived indices of metabolism exhibited by the cell line with a mis-splicing mutation (patient 6) confounds simple statistical comparisons of E1deWcient cells. It is noteworthy that NMR-derived indices of metabolism on patient 6 do not agree with the other E1-deWcient patient lines. This patient initially presented with delayed motor development and slightly elevated blood lactate levels. After diagnosis, he responded to a ketogenic diet, and at age 6 functioned as a 3–4 year old. This patient has an unusual manifestation of the disease, in that the splicing error in exon 6 of the sequence coding for the E1 subunit precludes its expression. However, the cells of this patient (the only male in this study), demonstrated somatic mosaicism [31]. It is possible that diVerences of the NMR-derived indices of metabolism between this patient and the others reXect the mild phenotype and presence of normal mitochondria in some cells obtained from this patient. DCA stimulates PDC activity principally by inhibiting PDC kinases that reversibly phosphorylate, and inactivate, the E1 subunit [32]. This eVect is rapid, and would be expected to occur shortly after exposing cells to DCA in vitro. In addition, studies in rats [33] and in cultured Wbroblasts from a few patients with PDC deWciency [34,35] indicate that a second, more protracted eVect of DCA is to stabilize E1 and decrease the ratio of turnover of the protein. Such a mechanism has been invoked to explain both the prolonged pharmacological eVects of the drug following its administration to humans [36,37] and its potential to aVect residual enzyme activity in cells from patients with PDC deWciency, due to mutations that lead to the synthesis of unstable E1 proteins. Thus, it is perhaps not surprising that the eVects of DCA we observed were qualitatively similar among both normal and PDC-deWcient cells and that these eVects were independent of the duration of the cells’ exposure to the drug. Such Wndings would be consistent with the primary action of DCA on the phosphorylation state of E1. DCA had little eVect on glycolysis in normal cells or on the overall bioenergetic status of either cell type. Its major inXuence was to shift entry of carbon into the TCA cycle from anaplerotic (PC) to oxidative (PDC) pathways. Additionally, isotopomeric analysis also demonstrated that DCA diversiWed the intramitochondrial sources for acetyl-CoA used by the TCA cycle. This

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suggests that it may have stimulated the oxidation of amino acids, fatty acids or both. ConWrmation of this notion will require additional studies using 13C isotopes of those substrates. In summary, NMR spectroscopy can elucidate pathways of intermediary, and particularly, intra-mitochondrial metabolism in non-transformed cultured human Wbroblasts. This technique appears useful for investigating the biochemical consequences of inborn errors of mitochondrial metabolism and the eVects of pharmacological agents thereon. Expansion of this approach may lead towards identifying the eVect of putative therapeutic agents for aZicted individuals, allowing for the development of a tailored therapy.

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