Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier

Abstract

Facilitated pyruvate transport across the mitochondrial inner membrane is a critical step in carbohydrate, amino acid, and lipid metabolism. We report that clinically relevant concentrations of thiazolidinediones (TZDs), a widely used class of insulin sensitizers, acutely and specifically inhibit mitochondrial pyruvate carrier (MPC) activity in a variety of cell types. Respiratory inhibition was overcome with methyl pyruvate, localizing the effect to facilitated pyruvate transport, and knockdown of either paralog, MPC1 or MPC2, decreased the EC₅₀ for respiratory inhibition by TZDs. Acute MPC inhibition significantly enhanced glucose uptake in human skeletal muscle myocytes after 2 h. These data (i) report that clinically used TZDs inhibit the MPC, (ii) validate that MPC1 and MPC2 are obligatory components of facilitated pyruvate transport in mammalian cells, (iii) indicate that the acute effect of TZDs may be related to insulin sensitization, and (iv) establish mitochondrial pyruvate uptake as a potential therapeutic target for diseases rooted in metabolic dysfunction.

Pyruvate uptake across the mitochondrial inner membrane is a central branch point in cellular energy metabolism with the ability to balance glycolysis and oxidative phosphorylation and poise catabolic and anabolic metabolism (1). Although the existence of a mitochondrial pyruvate carrier has been recognized for over 40 y (2, 3), it has only recently been identified at the molecular level. Two small transmembrane proteins in the inner membrane, mitochondrial pyruvate carrier 1 (MPC1) and 2 (MPC2), are obligate components of an apparent complex that facilitates inhibitor-sensitive pyruvate transport (4, 5). This newly defined complex may be a rational therapeutic target for modulating energy balance and the metabolic profile.

Thiazolidinediones (TZDs) are the most effective agents for preventing the progression from hyperglycemia to type 2 diabetes (6), and the increasingly appreciated link between dysregulated glucose metabolism and human disease has triggered the repurposing of pioglitazone to treat neurodegenerative conditions and certain cancers (7–9). However, significant side effects of TZDs, including volume expansion, bone loss, increased adiposity, and cardiovascular risk, have restricted broader clinical use (10, 11).

TZD activity is ascribed to peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor controlling gene expression related to lipid storage, cell differentiation, and inflammation (12, 13). However, a growing volume of data suggests that PPARγ-independent mechanisms, some of which are too rapid to be attributed to transcriptional events, may be relevant to effects on metabolism (14–20). In addition, the discovery that TZDs bind to mitochondrial membranes with low micromolar affinity (20), mirroring the circulating concentrations in treated patients (21, 22), suggests that some of their metabolic effects may be triggered by directly altering mitochondrial function.

This report demonstrates that TZDs specifically inhibit MPC activity. This inhibition can improve cellular glucose handling, as demonstrated by the finding that both TZDs and UK5099 [2-Cyano-3-(1-phenyl-1H-indol-3-yl)-2-propenoic acid], a chemical inhibitor of the MPC, rapidly increased glucose uptake in human myocytes. This discovery sets a precedent that pharmacologic targeting of the MPC can adjust cellular metabolism.

Results

Selective Permeabilization of the Plasma Membrane with Recombinant Perfringolysin O.

Although TZDs inhibit respiratory complex I at supraphysiological concentrations (23, 24), a direct effect on mitochondrial function from clinically relevant concentrations remains to be identified. To date, the ability to interrogate specific oxidative pathways with high-throughput respirometry has been limited by the lack of a reagent that permeabilizes the plasma membranes of cells in an adherent monolayer but is not injurious to mitochondria. Historically, various natural agents with differing mechanisms of action, including cytolysins (e.g., α-toxin of Staphylococcus aureus and streptolysin-O) and amphipathic glycosides (saponin and digitonin), have been used for cell permeabilization (25). In the present work, recombinant perfringolysin O (rPFO) (Materials and Methods) was used to selectively permeabilize the plasma membrane to control substrate provision.

rPFO is a recombinant, mutant form of a cholesterol-dependent cytolysin derived from Clostridium perfringens (26, 27). It oligomerizes to form pores that selectively permeabilize cellular plasma membranes and allow passage of solutes and large proteins of 200 kDa (28). Succinate and ADP, both of which are impermeable to the plasma membrane, sharply increased the rate of respiration in C2C12 myoblasts when acutely added with 1 nM rPFO (Fig. 1A). This concentration was sufficient to permeabilize 10 different cell types, including cell lines and primary cultures (Fig. S1A).

Fig. 1.

Selective permeabilization of the plasma membrane enables control of substrate provision and high-throughput respirometry. (A) C2C12 myoblasts were offered 15 mM glucose in MAS buffer [with 0.2% (wt/vol) BSA] and, where indicated, also offered 10 mM succinate, 4 mM ADP, and increasing concentrations of rPFO (n = 4). (B) Trace: Membrane potential of rat liver mitochondria (1 mg/mL) was monitored as described in SI Materials and Methods with safranine O. Additions were 1 μg/mL oligomycin, 10 nM rPFO, and successive additions of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) at 0.5 μM, 0.5 μM, and 1.0 μM. (Inset) Rat liver mitochondria in KCl-based buffer were treated with rPFO or digitonin for 5 min. and then centrifuged at 21,000 × g for 1 min at 4 °C. The release of cytochrome c from the intermembrane space to the supernatant (Sup.) or retained in the mitochondrial pellet was measured by Western blot analysis (SI Materials and Methods).

Unlike detergent-based methods, rPFO was not injurious to mitochondria. A 10-fold excess concentration of rPFO affected neither mitochondrial membrane potential nor cytochrome c release from the intermembrane space, a characteristic of digitonin (25) (Fig. 1B). rPFO also had no effect on state 3 respiration in isolated mitochondria, whereas increasing concentrations of digitonin caused a sharp decline (Fig. S1B). When added to cells, digitonin caused a drop in respiration that was rescued with exogenous cytochrome c, whereas respiration was again unchanged by rPFO (Fig. S1C).

TZDs Compromise Pyruvate-Driven Respiration in Permeabilized Cells.

In a variety of rPFO-permeabilized cells, TZDs inhibited pyruvate-dependent respiration (Figs. 2 and 3). In C2C12 myoblasts, rosiglitazone caused a dose-dependent decrease in pyruvate-driven, uncoupler-stimulated respiration (Fig. 2A). The effect occurred within minutes (making it unlikely to be mediated by transcriptional events) and was mimicked by UK5099, a potent inhibitor of the mitochondrial pyruvate transporter (29). Both rosiglitazone and troglitazone, a TZD withdrawn from clinical use due to hepatotoxicity (30), had half-maximal inhibitory concentrations (Fig. 2B) that approximate blood concentrations reached by TZDs in treated patients (21, 22). Metabolic Solutions Development Company (MSDC)-0160, a TZD structurally similar to pioglitazone (Fig. S2) and developed analogously to MSDC-0602 (20), had similar effects (Fig. 2B). This compound exhibits decreased apparent affinity to PPARγ (MSDC-0160, EC₅₀ = 23.7 μM; pioglitazone, EC₅₀ = 1.2 μM) while retaining binding to mitochondrial membranes (MSDC-0160, IC₅₀ = 1.3 μM; pioglitazone, IC₅₀ = 1.2 μM) (20). It has completed a promising Phase 2b trial for type 2 diabetes (31), and is currently being evaluated in an ongoing trial for mild cognitive impairment (ClinicalTrials.gov; NCT01374438).

Fig. 2.

TZDs inhibit pyruvate-driven respiration. (A) Representative experiment of permeabilized C2C12 myoblasts offered 5 mM pyruvate, 0.5 mM malate, 2 mM dichloroacetate (DCA), and 2 μM oligomycin in MAS buffer. Either rosiglitazone or UK5099 was added acutely where indicated, as was 400 nM FCCP. Error bars from technical replicates are obscured by the symbol. (B) Uncoupler-stimulated respiration with various TZDs was measured as in A; half-maximal inhibitory concentrations are given in parentheses (n ≥ 4). (C) Inhibition of pyruvate-driven respiration by MSDC-0160 in permeabilized primary cells was measured as in A with the following FCCP concentrations: 600 nM for NRVMs and HSkMMs, 300 nM for brown adipose tissue (BAT) precursors, and 250 nM for cortical neurons. Half-maximal inhibitory concentrations are given in parentheses (n ≥ 3). (D) Intact C2C12 myoblasts were offered pyruvate in unbuffered DMEM, and 10 μM MSDC-0160 or pioglitazone were added before 400 nM FCCP at the indicated times. MSDC-1473, a TZD negative control (EC₅₀ = 52 μM for PPARγ and >25 μM for mitochondrial membranes) (Fig. S2) was added 90 min before the addition of FCCP (n ≥ 3). n.s., not significant. (E) Intact HSkMMs were treated with TZDs (10 μM) or UK5099 (2 μM) at 90 min before the addition of 600 nM FCCP. When added acutely, 10 μM pioglitazone was added at 15 min before the addition of FCCP.

Fig. 3.

TZDs specifically inhibit mitochondrial pyruvate transport. (A) Uncoupler-stimulated respiration was measured acutely in permeabilized C2C12 myoblasts as in (Fig. 2A), but cells were offered either 10 mM glutamate + 5 mM malate or 10 mM succinate + 2 μM rotenone. The TZD concentrations were 3 μM, 10 μM, and 30 μM, and the corresponding UK5099 concentrations were 300 nM, 3 μM, and 10 μM (n = 4). (B) HSkMM, NRVMs, and cortical neurons were permeabilized and offered respiratory substrates, after which maximal FCCP-stimulated respiration (oligomycin present) was measured. Either 10 μM MSDC-0160 or 300 nM UK5099 was added 6 min before the addition of FCCP. Pyr., 5 mM pyruvate + 0.5 mM malate + 2 mM DCA; Glu., 10 mM glutamate + 5 mM malate; Succ., 10 mM succinate + 2 μM rotenone (n = 4). (C) FCCP-stimulated respiration was measured in HSkMMs and cortical neurons as in B. MSDC-0160 and rosiglitazone, 10 μM; troglitazone, 5 μM; UK5099, 300 nM. Pyr/Mal, 5 mM pyruvate + 0.5 mM malate + 2 mM DCA; MePyr + P/M, 20 mM methyl pyruvate + 5 mM pyruvate + 0.5 mM malate + 2 mM DCA. ^‡Significant rescue compared with matched treatment without methyl pyruvate (P < 0.05); ^‡‡P < 0.01. (D) Intact C2C12 myoblasts were given either 10 μM TZD or 2 μM UK5099. After 90 min, cells were washed and permeabilized, and then offered pyruvate, malate, DCA, oligomycin, and FCCP as before.

Importantly, the extent of inhibition by higher concentrations of TZDs matched that seen with UK5099 (Fig. 2B). The residual respiration was likely driven by passive entry of pyruvate, a weak acid, across the inner membrane (1). TZD inhibition of pyruvate oxidation was also apparent in four primary cell types. In human skeletal muscle myotubes (HSkMMs) obtained from quadriceps punch biopsies, neonatal rat ventricular myocytes (NRVMs), rat cortical neurons, and mouse brown adipocyte precursors, MSDC-0160 inhibited pyruvate-driven respiration with similar half-maximal concentrations (Fig. 2C), suggesting that the effect is cell type-independent.

Pioglitazone did not induce acute respiratory inhibition in permeabilized myoblasts (Fig. 2B); however, in intact myocytes, both immortalized (Fig. 2D) and primary (Fig. 2E), pioglitazone caused time-dependent respiratory inhibition. Given the immediate respiratory inhibition with MSDC-0160 (Fig. 2D), and the fact that pioglitazone is readily metabolized (21), it is possible that intact cells generate an active pioglitazone metabolite. Taken together, our data show that physiologically relevant concentrations of TZDs inhibit pyruvate oxidation.

Pyruvate Transport Is Specifically Compromised.

To determine the precise mechanism of respiratory inhibition, cells were permeabilized and offered different oxidizable substrates (Fig. S3). In permeabilized C2C12 myoblasts, TZDs significantly inhibited respiration driven by glutamate (with malate) only at 30 μM (Fig. 3A). Succinate-driven respiration was unaffected. These results are consistent with previous reports of complex I inhibition at supraphysiological TZD concentrations (23, 24). In three types of primary cells (HSkMMs, NRVMs, and cortical neurons), pyruvate-driven respiration was significantly compromised with both UK5099 and MSDC-0160, but there was no effect on the oxidation of either glutamate or succinate (Fig. 3B). This pinpoints the TZD effect at lower, clinically relevant concentrations to inhibition of either pyruvate transport or pyruvate dehydrogenase (PDH) activity.

To distinguish between these two mechanisms, patient-derived myotubes and cortical neurons were offered excess methyl pyruvate, which freely crosses the inner membrane and is cleaved by matrix esterases to generate intramitochondrial pyruvate (Fig. S3). On the addition of methyl pyruvate, the inhibitory effects of TZDs and UK5099 were almost entirely overcome. This indicates that TZDs are acute, specific inhibitors of mitochondrial pyruvate transport (Fig. 3C). However, unlike UK5099, which covalently modifies the pyruvate transporter via a reactive thiol group (32), the inhibitory effect of TZDs was reversible. After treatment of intact cells with TZDs, rates of pyruvate oxidation were restored on washing and permeabilization, whereas the effect of UK5099 persisted (Fig. 3D).

The MPC Complex Is a Target of TZDs.

Both MPC1 and MPC2 are requisite components of facilitated mitochondrial pyruvate uptake (4, 5). Each paralog was stably repressed in C2C12 myoblasts using lentiviral shRNA. RT-PCR (Fig. S4A) and Western blot analysis (Fig. S4B) confirmed knockdown of transcript and protein expression, and suggested that MPC1 and MPC2 are coordinately regulated. The functional consequence of this knockdown of either paralog was severely compromised pyruvate oxidation (Fig. 4A). The depressed respiration could not be attributed to differences in cell number, given that total cellular protein at the time of the assay was equivalent in all lines (Fig. S4C). Moreover, respiration on complex I-linked substrates (glutamate and β-hydroxybutyrate), Q-pool substrates (succinate and glycerol-3-phosphate), or palmitoyl carnitine was unchanged with stable repression of either MPC paralog in C2C12 myoblasts (Fig. 4B). As before, methyl pyruvate could significantly rescue respiration in cell lines with knockdown of either MPC paralog (Fig. 4C), indicating that repressed expression did not cause global mitochondrial dysfunction.

Fig. 4.

Knockdown of MPC1 and MPC2 specifically compromises pyruvate oxidation and increases sensitivity to MPC inhibitors. (A) A representative experiment in which permeabilized, transduced cells were offered 5 mM pyruvate (with malate, DCA, and oligomycin as before) followed by 400 nM FCCP. UK5099, 300 nM. Where not visible, error bars from technical replicates are obscured by the symbol. OCR, oxygen consumption rate. (B) Uncoupler-stimulated respiration was measured in permeabilized, transduced cells offered different oxidizable substrates. Abbreviations and concentrations are as in Fig. 3B. Palm, 40 μM palmitoyl carnitine + 0.5 mM malate. β-OH, 10 mM β-hydroxybutyrate + 0.5 mM malate; G-3-P, 10 mM glycerol-3-phosphate + 2 μM rotenone. (C) Uncoupler-stimulated respiration was measured as in A, with abbreviations as in Fig. 3C. ^‡Significant rescue relative to matched treatment without methyl pyruvate, P < 0.05; ^‡‡P < 0.01. (D) Concentration-response curves of pyruvate-driven, uncoupler-stimulated respiration were generated with permeabilized, transduced cells. An aggregate curve of six biological replicates acutely given MSDC-0160 is presented. Substrate and uncoupler concentrations are as in Fig. 2A. (E) EC₅₀ values for UK5099 and MSDC-0160 inhibition were calculated for each replicate experiment (n = 6).

If indeed the MPC complex is a target of TZDs, then knockdown should reduce the EC₅₀ necessary to inhibit pyruvate-driven respiration. This was true for MSDC-0160 (Fig. 4 D and E) and for UK5099 (Fig. 4E), a crucial positive control. This result strongly suggests that the MPC complex is a target of TZDs at clinically relevant concentrations.

Partial MPC Inhibition Increases Cellular Glucose Uptake.

Both in intact muscle (16) and in vivo (33), TZDs can acutely increase glucose uptake on a scale that suggests that their enhanced transport activity might not be entirely explained by transcriptional events (≤30 min in skeletal muscle, 120 min in vivo). To determine whether mild MPC inhibition can account for this, we measured glucose uptake in L6 myotubes (Fig. 5A) and HSkMMs (Fig. 5B). After 90–120 min, both pioglitazone (10 μM; Fig. 5A) and troglitazone (11 μM; Fig. 5B), like insulin, significantly increased plasma membrane glucose uptake. To link this effect to MPC inhibition, we also measured uptake in response to UK5099 in both cell types (Fig. 5C, blue). The degree to which glucose uptake was stimulated in either cell type was directly proportional to the extent of respiratory inhibition by TZDs or UK5099 (Fig. 5C). Consistent with previous reports (34–36), treatment of patient myotubes with 10 μM pioglitazone increased phosphorylation of AMP-activated protein kinase (AMPK) (Fig. 5D). Again, this acute effect of TZDs was mimicked with UK5099, linking changes in MPC activity with cytoplasmic energy sensing.

Fig. 5.

Mild MPC inhibition increases plasma membrane glucose uptake and activates AMPK. (A) Glucose uptake was measured in L6 myotubes after a 2-h treatment with either 32 nM insulin or 10 μM pioglitazone (n = 6). (B) Glucose uptake was measured in HSkMMs after a 90-min treatment with either 32 nM insulin or 11 μM troglitazone (n = 12). (C) The increase in the rate of glucose uptake is plotted against the degree of respiratory inhibition for each treatment. Respiration was measured in intact L6 myotubes (circles) and HSkMMs (triangles) as in Fig. 2E. Gray, basal (no drug added); blue, 2 μM UK5099; red, 10 μM pioglitazone; purple, 11 μM troglitazone; broken line, regression analysis (r² = 0.92). (D) HSkMMs were treated for 2 h with either 10 μM pioglitazone or 2 μM UK5099 as in Fig. 5A, and prepared for Western blot analysis. (Upper) Sample immunoblots of pAMPK and AMPK from matched samples. (Lower) Densitometry analysis from four different patient samples.

Discussion

This study provides unequivocal evidence that TZDs are acute, specific inhibitors of the mitochondrial pyruvate carrier at clinically relevant concentrations. In addition to demonstrating that the recently defined MPC is a target of these effective insulin sensitizers, it also provides evidence that acute inhibition of MPC activity can regulate cellular glucose metabolism. Of course, these data do not obviate involvement of PPARγ in the insulin-sensitizing effects of TZDs. Rather, we suggest that TZDs work through a previously undefined, pleiotropic mechanism in which both transcriptional regulation and acute MPC inhibition enhance the metabolic profile.

This work was empowered by the use of rPFO-permeabilized cells to measure mitochondrial respiration in situ. Cells permeabilized with rPFO were not subject to the drawbacks associated with detergent-based methods, such as mitochondrial outer membrane damage and cell detachment, possibly attributable to the threshold cholesterol content required for pore formation (37). Such reliability allowed us to perform a mechanistic analysis despite a limited sample size in primary cells, and gave us the ability to interrogate mitochondrial function in genetically modified cells without isolation-induced mitochondrial damage.

Rigorous bioenergetic analysis coupled with genetic suppression of either obligatory MPC paralog demonstrated that TZDs specifically inhibited mitochondrial pyruvate uptake. The effect occurred within minutes in permeabilized cells, rendering transcriptional activation an unlikely mechanism, and at single-digit micromolar concentrations that reflect circulating levels in treated patients (21, 22). Although previous work documented that TZDs can inhibit complex I (23, 24), the concentrations used exceeded physiological relevance; indeed, some TZDs in the present study significantly inhibited complex I at 30 μM. One preliminary report has suggested that TZDs exert substrate-specific effects on respiration of isolated brain mitochondria (15), but an in-depth mechanistic analysis was not reported. MitoNEET has also been put forth as a direct mitochondrial target of TZDs (38), but to date, there are no data showing that altered protein expression can modulate TZD binding or efficacy.

Our work can also further define the explicit function of MPC1 and MPC2. Questions have been raised about whether existing evidence can discriminate between a distinct role for the MPC complex in pyruvate transport as opposed to a separate role in overall pyruvate metabolism (39). Although flux through the PDH complex certainly can affect the rate of pyruvate transport, the demonstration that excess methyl pyruvate can almost entirely rescue respiration from both MPC inhibition and knockdown indicates that the function of the MPC complex is discrete from PDH activity. Furthermore, the yeast glycerol-3-phosphate dehydrogenase was reported to physically associate with the MPC complex (5). However, we found that the stable knockdown of either paralog had no effect on respiration rates in permeabilized C2C12 myoblasts oxidizing glycerol-3-phosphate (Fig. 4B).

Although it may seem counterintuitive that reduced mitochondrial pyruvate uptake may be a mechanism of insulin sensitization, it is important to note that the restriction of pyruvate-driven respiration by TZDs is never complete and is readily reversible (Figs. 2 and 3D). Clinically relevant drug concentrations cannot entirely block pyruvate transport and oxidation, which presumably would be a toxic effect inconsistent with the clinical utility of TZDs. Moreover, unlike inhibition by UK5099, the reversibility of the interaction may induce a conditioning effect whereby mild, transient MPC inhibition potentiates reliance on alternative substrates. Although the formal possibility exists that this interaction mediates toxicity, the demonstration that MPC inhibition by TZDs can acutely increase glucose uptake and increase AMPK phosphorylation proves the principle that partial MPC inhibition can acutely improve cellular glucose handling.

Substantial inhibition of pyruvate entry into the matrix might be expected to increase lactate levels (40). However, unlike metformin treatment, lactic acidosis has not generally been reported as a consequence of chronic TZD administration (41), suggesting a tonic degree of MPC inhibition in treated patients. It is crucial to note that MPC inhibition still allows the oxidation of amino acids, fatty acids, and other complex I-linked substrates (Fig. 4B and Fig. S3).

Many of the beneficial effects of TZDs on whole-body metabolism may, to some degree, be attributable to MPC inhibition as well. Restricted mitochondrial pyruvate uptake might suppress flux through pyruvate carboxylase, limiting the fuel available for hepatic gluconeogenesis (42). This mechanism also might help explain why TZDs can decrease lipid accumulation in the liver and skeletal muscle (43, 44). MPC inhibition likely would diminish the pool of intramitochondrial citrate, potentially reducing its efflux and, in turn, lipogenesis. If so, then the associated production of malonyl CoA would decrease as well. This would relieve malonyl CoA-mediated inhibition of carnitine palmitoyl transferase I and accelerate fatty acid oxidation, a characteristic of skeletal muscle myocytes exposed to chronic TZD treatment (35, 45, 46). Furthermore, reduced intramitochondrial pyruvate likely would enhance amino acid oxidation to maintain tricarboxylic acid cycle activity and ATP production. It also may stimulate mitochondrial malic enzyme activity, producing pyruvate from malate and hence enhancing NAD(P)H levels.

Perhaps the strongest evidence that mild MPC inhibition can be insulin-sensitizing is the increase in glucose uptake observed in L6 myotubes and HSkMMs. Enhanced glucose transport occurred within 90 min of TZD treatment in patient-derived myotubes, and could be reproduced by the MPC inhibitor UK5099. Previous work has in fact reported that 30 μM TZD enhanced the rate of glucose metabolism in rat cortical astrocytes (47), although this concentration can cause respiratory inhibition of complex I. Although others have noted that TZD administration can acutely activate AMPK (34–36) and subsequently stimulate glucose uptake through a PPARγ-independent mechanism (16), this report demonstrates that these effects can be reproduced with UK5099 (Fig. 5C), a specific inhibitor of the MPC (Fig. 3A). MPC inhibition also may trigger signaling via protein acetylation on either side of the mitochondrial inner membrane, given that impaired mitochondrial pyruvate uptake would increase the concentration of pyruvate, and thus of acetyl units, in the cytoplasm. Acetylation as a posttranslational modification is likely important to the regulation of cell metabolism (48).

We propose that mild inhibition of pyruvate transport by TZDs induces a beneficial, hormetic effect on whole-body metabolism. This mechanism can potentially explain their acute insulin-sensitizing effects by initiating a cascade of events including increased glucose uptake and enhanced oxidation of alternative fuels, such as fatty and amino acids. Modulation of this insulin-independent mechanism, potentially mediated by AMPK, could be of tremendous benefit for the treatment of metabolic syndrome and type 2 diabetes (49, 50). Moreover, dysregulated glucose metabolism occurs not only in type 2 diabetes, but also in pathologies, including cancer, neurodegenerative disease, and heart failure. As such, the demonstration that pharmacologic modulation of MPC can regulate the pattern of cellular glucose metabolism establishes an important avenue for drug development centered around this target.

Materials and Methods

Animals and Human Subjects.

All animal protocols were approved by the University of California at San Diego’s Institutional Animal Care and Use Committee. Human skeletal muscle biopsy specimens were obtained from subjects with approval of the University of California at San Diego’s Committee on Human Investigation. Informed written consent for biopsy was obtained from all subjects after explanation of the protocol. Samples were obtained from those who displayed normal glucose tolerance in response to a standard 75-g oral glucose tolerance test and lacked a familial history of type 2 diabetes.

Cell Culture.

C2C12 mouse and L6 rat myoblasts were obtained from American Type Culture Collection and cultured as suggested by the supplier. Myocytes from human skeletal muscle biopsy specimens were prepared as described previously (51). NRVMs were prepared according to published methods (52). After isolation, cell suspensions were preplated for 2 h to reduce fibroblast contamination. Murine brown adipocyte tissue precursors were prepared as described previously using CD1 mice (21–28 d old) (53). Rat cortical neurons were prepared from E18 Sprague–Dawley rats according to published methods (54).

Mitochondrial Isolation, Membrane Potential Measurements, and Cytochrome c Release.

Mitochondria from rat skeletal muscle, rat liver, and C2C12 cells were isolated by differential centrifugation (55). Rat liver mitochondrial membrane potential was monitored with 5 μM safranine O at 495 nm excitation/586 nm emission. Cytochrome c release was measured in supernatants and pellets from incubations of rat liver mitochondria in KCl-based medium, as described in SI Materials and Methods.

Respirometry.

Respiration in intact and permeabilized cells was measured using a Seahorse XF24 or XF96 Extracellular Flux Analyzer (Seahorse Bioscience). Unless specified otherwise, intact cells were offered 10 mM glucose, 10 mM Na⁺ pyruvate, and 2 mM GlutaMAX (Invitrogen) in unbuffered DMEM (D5030; Sigma-Aldrich), pH 7.4, at 37 °C.

Respirometry with permeabilized cells was conducted in MAS buffer (70 mM sucrose, 220 mM mannitol, 10 mM KH₂PO₄, 5 mM MgCl₂, 2 mM Hepes, and 1 mM EGTA; pH 7.2) at 37 °C without BSA unless stated otherwise. rPFO [XF Plasma Membrane Permeabilizer (XF PMP); Seahorse Bioscience] is a recombinant perfringolysin O derivative (PFO^C459A) that requires a higher threshold level of cholesterol than native PFO (37), optimal for selective plasma membrane permeabilization. rPFO was added at 1 nM to selectively permeabilize the plasma membrane. The ATP synthase inhibitor oligomycin (2 μM) and oxidizable substrates were provided as indicated.

Lentiviral shRNA Knockdown, RT-PCR, and Western Blot Analysis.

Cells with stable repression of MPC1 and MPC2 were generated with MISSION lentiviral shRNA plasmids under puromycin selection. Cells were lysed, and mRNA was extracted using the RNeasy Kit (Qiagen) with on-column DNase digestion (Qiagen). Mitochondrial protein and cell lysates were solubilized and run on a Laemmli gel, transferred to PVDF, and analyzed by immunoblotting for MPC1, MPC2, cytochrome c, AMPK, and phosphorylated AMPK (pAMPK).

Glucose Uptake.

Glucose uptake was measured as described previously (56). Differentiated L6 or patient derived myocytes were washed in serum-free medium and incubated ± insulin (32 nM) or drug (10 μM TZD or 2 μM UK5099) for 90 min at 37 °C in a 5% (vol/vol) CO₂ incubator. Glucose uptake, quantified using the nonmetabolized radiolabeled analog 2-deoxyglucose (10 μM final concentration), was measured in triplicate over 10 min at room temperature. Data were normalized to the protein content in each well. The uptake of labeled L-glucose was used to correct samples for the nonspecific diffusion of tracer.

Statistics.

Statistical analysis and curve fitting were conducted using GraphPad Prism. Significance was assessed by ANOVA for repeated measures with Dunnett’s posttest (95% confidence interval). When data were expressed as a percentage of control values, significance was calculated on the square root of the normalized data. A P value < 0.05 (*) was considered statistically significant (**P < 0.01; ***P < 0.001). Data are presented as mean ± SEM.

Note Added in Proof.

While this report was in press, the observation that initiated this study, demonstrating that thiazolidinediones can directly bind a protein complex containing MPC2, was accepted for publication (57).