Regulation of Substrate Utilization by the Mitochondrial Pyruvate Carrier

Reliance on substrates for energy and biosynthetic metabolism shifted from glucose to amino acid and fatty acid oxidation upon MPC inhibition.

“Surprisingly, proliferating myoblasts maintained growth and ATP-linked respiration despite profound inhibition of MPC activity; however, reliance on substrates for energy and biosynthetic metabolism shifted from glucose to amino acid and fatty acid oxidation.”

Oxidative TCA flux was achieved through enhanced reliance on glutaminolysis through malic enzyme and pyruvate dehydrogenase (PDH) as well as fatty acid and branched chain amino acid oxidation.

“Oxidative TCA flux was achieved through enhanced reliance on glutaminolysis through malic enzyme and pyruvate dehydrogenase (PDH) as well as fatty acid and branched chain amino acid oxidation.”

The extent of glutamine conversion to the lipogenic AcCoA pool was significantly increased in Mpc2KD cells.

“Although we observed no significant change in relative palmitate synthesis upon Mpc depletion (Figure 2J), the extent of glutamine conversion to the lipogenic AcCoA pool was significantly increased in Mpc2KD cells (Figure 2K).”

The abundance of glutamine-derived alanine was significantly elevated in Mpc1/2KD cells.

“Furthermore, the abundance of glutamine-derived alanine was significantly elevated (Figure 2H), consistent with higher flux through mitochondrial MEs as opposed to reductive carboxylation.”

Pharmacological inhibition of MPC activity in hSKMs increased the extent that branched chain amino acids (BCAAs) were oxidized in the TCA cycle.

“Pharmacological inhibition of MPC activity in hSKMs increased the extent that branched chain amino acids (BCAAs) were oxidized in the TCA cycle.”

TCA flux and fatty acid synthesis were maintained through increased glutamine anaplerosis and oxidation, malic enzyme flux, and fatty acid oxidation.

“TCA flux and fatty acid synthesis were maintained through increased glutamine anaplerosis and oxidation, malic enzyme flux, and fatty acid oxidation.”

Proliferating myoblasts maintained growth and ATP-linked respiration despite profound inhibition of MPC activity.

“Surprisingly, proliferating myoblasts maintained growth and ATP-linked respiration despite profound inhibition of MPC activity; however, reliance on substrates for energy and biosynthetic metabolism shifted from glucose to amino acid and fatty acid oxidation.”

The relative abundances of both M5 and M6 citrate mass isotopomers from [U-13C5]glutamine were significantly increased in Mpc1/2KD cells.

“The relative abundances of both M5 and M6 mass isotopomers were significantly increased in Mpc1/2KD cells (Figure 2G).”

No significant change in relative palmitate synthesis was observed upon Mpc depletion.

“Although we observed no significant change in relative palmitate synthesis upon Mpc depletion (Figure 2J), the extent of glutamine conversion to the lipogenic AcCoA pool was significantly increased in Mpc2KD cells (Figure 2K).”

De novo serine synthesis from glucose in the cytosol was increased in Mpc1/2KD cells.

“Additionally, we observed an increase in de novo serine synthesis from glucose in the cytosol (Figure 2D), highlighting potential compartment-specific shifts in amino acid metabolism (Figure S1B).”

Labeling of citrate and all TCA intermediates from [U-13C6]glucose was significantly decreased in Mpc1/2KD cells relative to controls.

“Consistent with the expected decrease in glucose-derived pyruvate oxidation, labeling of citrate and all TCA intermediates was significantly decreased in Mpc1/2KD cells relative to controls (Figure 2A-2B).”

Uncoupler-stimulated respiration in Mpc1/2KD cells was more sensitive to BPTES treatment.

“Uncoupler-stimulated respiration in Mpc1/2KD cells was more sensitive to BPTES treatment, signifying an increased reliance on glutamine oxidation in these cells (Figure 2I).”

Citrate levels were markedly decreased in Mpc1KD or Mpc2KD (Mpc1/2KD) cells.

“Citrate levels, however, were markedly decreased in Mpc1KD or Mpc2KD (Mpc1/2KD) cells, as this metabolite is generated predominantly from pyruvate-derived AcCoA under normal growth conditions (Metallo et al., 2012).”

The abundance of fully labeled succinate, fumarate, α-ketoglutarate, and malate from [U-13C5]glutamine increased significantly in Mpc1/2KD cells.

“The abundance of fully labeled succinate, fumarate, α-ketoglutarate, and malate increased significantly in Mpc1/2KD cells (Figure 2E), suggesting that cells increase their reliance on glutamine anaplerosis when mitochondrial pyruvate transport is limited.”

C2C12 myoblasts displayed a shift away from glucose to glutamine as a substrate for fatty acid synthesis in the presence of UK5099.

“C2C12 myoblasts also displayed a shift away from glucose to glutamine as a substrate for fatty acid synthesis in the presence of UK5099 (Figure 4C).”

A significant increase in glutaminolysis was observed in C2C12 myotubes treated with UK5099, as evidenced by steady-state labeling from [U-13C5]glutamine.

“Additionally, we observed a significant increase in glutaminolysis in C2C12 myotubes treated with UK5099, as evidenced by steady-state labeling from [U-13C5]glutamine (Figure 5G).”

M2 labeling was significantly increased in Mpc2KD myoblasts cultured with [3-13C]glutamine.

“We also measured a significant increase in M2 labeling in Mpc2KD myoblasts cultured with [3-13C]glutamine (Figure 2M), which arises from condensation of labeled oxaloacetate and AcCoA (Figure S1C).”

Both A549 cells and the Huh7 human hepatocarcinoma cell line increased their reliance on fatty acid oxidation to fuel TCA metabolism upon MPC inhibition.

“Finally, both A549 cells and the Huh7 human hepatocarcinoma cell line increased their reliance on fatty acid oxidation to fuel TCA metabolism upon MPC inhibition (Figure S3L-S3M).”

Increased label incorporation into numerous TCA metabolites downstream of citrate was detected in Mpc2KD cells.

“Increased label incorporation into numerous TCA metabolites downstream of citrate was also detected (Figure 3C), indicating that knockdown of Mpc2 induced a significant increase in fatty acid oxidation flux in C2C12 myoblasts.”

Consistent increases in BCAA oxidation were observed in hSKMs obtained from 2 independent subjects.

“Consistent increases in BCAA oxidation were observed in hSKMs obtained from 2 independent subjects (Figures 5H and S4G).”

Knockdown of Mpc did not affect glycolytic flux in C2C12 myotubes, as lactate secretion, glucose uptake and the ratio of the two were unchanged.

“Knockdown of Mpc did not affect glycolytic flux in C2C12 myotubes, as lactate secretion, glucose uptake and the ratio of the two were surprisingly unchanged (Figure 5C).”

The relative abundance of M2 citrate from [U-13C16]palmitate was significantly increased in Mpc2KD compared to control cells.

“We observed a significant increase in the relative abundance of M2 citrate from this tracer in Mpc2KD compared to control cells (Figure 3B).”

Knockdown of either Mpc1 or Mpc2 abrogated expression of both proteins.

“Knockdown of either Mpc1 or Mpc2 abrogated expression of both proteins, as observed previously (Divakaruni et al., 2013).”

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In the presence of 10 mM extracellular pyruvate, Mpc2KD cells failed to increase the contribution of glutamine carbon to palmitate synthesis.

“In the presence 10 mM extracellular pyruvate, Mpc2KD cells failed to increase the contribution of glutamine carbon to palmitate synthesis.”

Glucose oxidation was inhibited in hSKMs cultured with UK5099.

“As expected, glucose oxidation was inhibited in hSKMs cultured with UK5099 (Figure 5I).”

Alanine levels were significantly decreased in Mpc knockdown cells.

“Notably, alanine levels were significantly decreased in Mpc knockdown cells, highlighting a role for mitochondrial pyruvate and mitochondrial alanine aminotransferase (ALT2) in mediating glutamine anaplerosis which was previously described in cancer cells (Weinberg et al., 2010).”

Glucose metabolism in the TCA cycle was significantly decreased but not completely in MPC knockdown cells.

“Glucose metabolism in the TCA cycle was significantly decreased but not completely, presumably due to incomplete knockdown, passive diffusion, or the presence of an alternate or non-facilitated transport mechanism.”

When cells were treated with combinations of UK5099, BPTES, and etomoxir, a significant effect of UK5099 was detected only with combinatorial treatments.

“However, when cells were treated with combinations of UK5099, BPTES, and etomoxir we detected a significant effect of UK5099 only with combinatorial treatments (Figures 4D, S2F-S2G for raw values).”

In contrast to proliferating cells, myotubes rely primarily on glucose carbon for the generation of TCA intermediates rather than glutamine.

“In contrast to proliferating cells, myotubes rely primarily on glucose carbon for the generation of TCA intermediates (rather than glutamine) (Figure S4C).”

A significant increase in BCAA oxidation was observed upon MPC inhibition using hSKM cultures.

“We also observed a significant increase in BCAA oxidation upon MPC inhibition using hSKM cultures, which are of perhaps greater physiological relevance than C2C12 cells.”

No significant flux through BCAA oxidation was detected in C2C12 myoblasts, myotubes, or engineered lines using [13C]BCAA tracers.

“We failed to detect significant flux through this pathway in any of the C2C12 myoblasts (Figure S2C), myotubes, or engineered lines generated here using [13C]BCAA tracers.”

The specific changes in amino acid and fatty acid metabolism were recapitulated using the MPC inhibitor UK5099.

“Although ATP-linked respiration and cell growth remained unchanged, we identified specific changes in amino acid and fatty acid metabolism that were recapitulated using the MPC inhibitor UK5099.”

Lack of branched chain amino acid (BCAA) oxidation to TCA intermediates was observed in both Control and Mpc2KD cells.

“Given the lack of branched chain amino acid (BCAA) oxidation to TCA intermediates observed in both Control and Mpc2KD cells (Figure S2C) we hypothesized that this AcCoA was derived from β-oxidation.”

Increased amino acid and fatty acid oxidation were observed upon inhibition of MPC.

“In addition to these metabolomic changes we also observed increased amino acid and fatty acid oxidation upon inhibition of MPC.”

Similar trends in glutaminolysis were observed in myotubes generated using Control or Mpc2KD cells.

“Similar trends were observed in myotubes generated using Control or Mpc2KD cells (Figure S4F).”

No change in ATP-linked or maximal respiration was observed with UK5099 treatment.

“We observed no change in ATP-linked or maximal respiration with UK5099 treatment, presumably due to the metabolic plasticity of cells when other pathways (i.e., glutamine and fatty acid oxidation) were not inhibited (Figures 4D, S2F-S2G for raw values).”

MPC1KD in A549 cells exhibited an increased reliance on fatty acid oxidation to fuel TCA cycle metabolism.

“Finally, we observed that MPC1KD in A549 cells exhibited an increased reliance on fatty acid oxidation to fuel TCA cycle metabolism, as 13C labeling of citrate and other TCA metabolites derived from [U-13C]palmitate increased significantly (Figures S3H-S3I).”

13C enrichment throughout the TCA cycle was significantly increased in hSKMs treated with 10 μM UK5099 compared to controls.

“Notably, 13C enrichment throughout the TCA cycle was significantly increased in hSKMs treated with 10 μM UK5099 compared to controls (Figures 5H and S4G).”

Glutamine anaplerosis and oxidation through malic enzymes were elevated upon MPC KD in A549 cells.

“On the other hand, glutamine anaplerosis and oxidation through malic enzymes were elevated upon MPC KD in these cells, as evidenced by labeling of TCA intermediates (citrate in particular) and alanine from [U-13C5]glutamine and [3-13C]glutamine (Figure S3D-S3G).”

Specific changes in amino acid and fatty acid metabolism were identified in MPC knockdown cells.

“Although ATP-linked respiration and cell growth remained unchanged, we identified specific changes in amino acid and fatty acid metabolism that were recapitulated using the MPC inhibitor UK5099.”

No change in labeling was observed in lactate in Mpc2KD myoblasts cultured with [3-13C]glutamine.

“No change in labeling was observed in lactate (Figure S1D), providing evidence that this ME flux was catalyzed within the mitochondrial compartment.”

Flux from the exogenous AcCoA pool into the TCA cycle increased 5-fold in Mpc2KD cells compared to Control cells.

“Flux from this AcCoA pool into the TCA cycle increased 5-fold in Mpc2KD cells compared to Control cells, suggesting that oxidation of mitochondrial substrates other than glutamine were also induced upon Mpc depletion (Figure 3A and Tables S1-S2).”

ATP-linked respiration was only affected when all three pathways (pyruvate transport, glutamine and fatty acid oxidation) were inhibited.

“ATP-linked respiration was only affected when all three pathways (i.e., pyruvate transport, glutamine and fatty acid oxidation) were inhibited (Figures 1D, 3D, and S2D for raw values).”

The increased glutamine-derived AcCoA in Mpc2KD cells was almost exclusively attributed to the glutaminolysis pathway.

“The increased glutamine-derived AcCoA was almost exclusively attributed to the glutaminolysis pathway, as evidenced by transfer of label from [3-13C]glutamine to palmitate (Figure 2L, S1C).”

Targeted metabolomic analysis revealed increases in intracellular abundances of pyruvate and aspartate in Mpc2KD myotubes.

“Targeted metabolomic analysis revealed increases in intracellular abundances of pyruvate and aspartate in Mpc2KD myotubes (Figure S4B).”

The difference in glutamine carbon contribution to palmitate synthesis in Mpc2KD cells with 10 mM pyruvate was almost entirely accounted for by conversion of exogenous [3-13C]pyruvate to lipogenic AcCoA through the PDH complex.

“Indeed, this difference was almost entirely accounted for by the conversion of exogenous [3-13C]pyruvate to lipogenic AcCoA through the PDH complex (Figure 2N).”

No increase in reductive carboxylation flux was observed upon Mpc knockdown.

“Surprisingly, we observed no increase in reductive carboxylation flux.”

Supplementation of pyruvate at concentrations expected to enter mitochondria through passive diffusion abrogated the increase in glutamine to fatty acid conversion.

“While our MFA results indicated that overall flux through PDH was decreased in Mpc2 knockdown cells, supplementation of pyruvate at concentrations expected to enter mitochondria through passive diffusion abrogated the increase in glutamine to fatty acid conversion.”

Mpc inhibition promoted oxidative glutaminolysis to fuel the mitochondrial AcCoA pool.

“Whereas elevated glycolytic rates and reductive carboxylation are predominant modes of metabolism under such conditions (Metallo et al., 2012; Mullen et al., 2012; Scott et al., 2011; Wise et al., 2011), Mpc inhibition instead promoted oxidative glutaminolysis to fuel the mitochondrial AcCoA pool.”

Overall flux through PDH was decreased in Mpc2 knockdown cells.

“While our MFA results indicated that overall flux through PDH was decreased in Mpc2 knockdown cells, supplementation of pyruvate at concentrations expected to enter mitochondria through passive diffusion abrogated the increase in glutamine to fatty acid conversion.”

Citrate mass isotopomer labeling and flux estimations highlighted a significant increase in flux through malic enzyme upon Mpc knockdown.

“Citrate mass isotopomer labeling and flux estimations also highlighted a significant increase in flux through malic enzyme upon Mpc knockdown, which could provide additional reducing equivalents [NAD(P)H] within the matrix.”

Fatty acid synthesis rates were maintained upon Mpc knockdown.

“Fatty acid synthesis rates were also maintained upon Mpc knockdown, in contrast to that observed when comparing normoxic and hypoxic cell growth (Kamphorst et al., 2013; Metallo et al., 2012).”

Conversion of [13C]pyruvate carbons to lipogenic AcCoA confirmed this pyruvate was metabolized by PDH.

“Furthermore, conversion of [13C]pyruvate carbons to lipogenic AcCoA confirmed this pyruvate was metabolized by PDH, since carbon atoms in oxaloacetate do not enter the AcCoA pool without PDH activity.”

Dysregulation of pyruvate metabolism contributes to the pathogenesis of numerous diseases, including diabetes and obesity, mitochondrial disorders, cardiac failure, neurodegenerative disorders, and cancer.

“Dysregulation of these processes contributes to the pathogenesis of numerous diseases, including diabetes and obesity (DeFronzo and Tripathy, 2009; Sugden et al., 2009), mitochondrial disorders (Kerr, 2013), cardiac failure (Fillmore and Lopaschuk, 2013), neurodegenerative disorders (Cunnane et al., 2011; Yao et al., 2011), and cancer (Currie et al., 2013; Tennant et al., 2010).”

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A protein carrier facilitates pyruvate transport into mitochondria.

“Existence of a protein carrier to facilitate pyruvate transport into mitochondria has been recognized for decades (Halestrap and Denton, 1974; Papa et al., 1971).”

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Higher flux through mitochondrial malic enzymes, as opposed to reductive carboxylation, occurs in Mpc1/2KD cells.

“Furthermore, the abundance of glutamine-derived alanine was significantly elevated (Figure 2H), consistent with higher flux through mitochondrial MEs as opposed to reductive carboxylation.”

Amino acid metabolism was altered to maintain metabolic homeostasis in Mpc1/2KD cells.

“Aspartate and alanine abundances were significantly increased and decreased, respectively, suggesting that amino acid metabolism was altered to maintain metabolic homeostasis.”

Suppression of pyruvate transport induces a form of metabolic flexibility associated with use of lipids and amino acids as catabolic and anabolic fuels, in contrast to inhibition of complex I or PDH.

“Thus, in contrast to inhibition of complex I or PDH, suppression of pyruvate transport induces a form of metabolic flexibility associated with use of lipids and amino acids as catabolic and anabolic fuels.”

Cells with depleted Mpc1 or Mpc2 increase oxidative glutamine metabolism to maintain flux through the TCA cycle.

“Collectively, these results provide evidence that cells with depleted Mpc1 or Mpc2 increase oxidative glutamine metabolism to maintain flux through the TCA cycle.”

The genes MPC1 and MPC2 encode the multimeric mitochondrial pyruvate carrier (MPC) complex embedded in the mitochondrial inner membrane.

“Two recent studies revealed strong evidence that the genes, renamed MPC1 and MPC2, encode the multimeric mitochondrial pyruvate carrier (MPC) complex embedded in the mitochondrial inner mitochondrial membrane (Bricker et al., 2012; Herzig et al., 2012).”

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Cells increase their reliance on glutamine anaplerosis when mitochondrial pyruvate transport is limited.

“The abundance of fully labeled succinate, fumarate, α-ketoglutarate, and malate increased significantly in Mpc1/2KD cells (Figure 2E), suggesting that cells increase their reliance on glutamine anaplerosis when mitochondrial pyruvate transport is limited.”

Stable knockdown of Mpc1 or Mpc2 was confirmed at the transcriptional and protein levels.

“Stable knockdown was confirmed at the transcriptional and protein levels (Figures 1A-1B).”

Mpc1/2KD cells have increased reliance on glutamine oxidation.

“Uncoupler-stimulated respiration in Mpc1/2KD cells was more sensitive to BPTES treatment, signifying an increased reliance on glutamine oxidation in these cells (Figure 2I).”

Compartment-specific shifts in amino acid metabolism occur upon Mpc depletion.

“Additionally, we observed an increase in de novo serine synthesis from glucose in the cytosol (Figure 2D), highlighting potential compartment-specific shifts in amino acid metabolism (Figure S1B).”

Citrate is generated predominantly from pyruvate-derived AcCoA under normal growth conditions.

“Citrate levels, however, were markedly decreased in Mpc1KD or Mpc2KD (Mpc1/2KD) cells, as this metabolite is generated predominantly from pyruvate-derived AcCoA under normal growth conditions (Metallo et al., 2012).”

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Coexpression of Mpc1 and Mpc2 in Lactococcus lactis induced a fourfold increase in pyruvate uptake.

“Indeed, Herzig et al. observed that coexpression of Mpc1 and Mpc2 in Lactococcus lactis induced a fourfold increase in pyruvate uptake (Herzig et al., 2012).”

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Steady state isotopic labeling was observed after 24 hours of [U-13C6]glucose culture.

“To gain more detailed insights into mitochondrial substrate utilization we cultured cells in the presence of [U-13C6]glucose for 24 hours and observed steady state isotopic labeling (Figure S1A).”

The malic enzyme flux in Mpc2KD cells was catalyzed within the mitochondrial compartment.

“No change in labeling was observed in lactate (Figure S1D), providing evidence that this ME flux was catalyzed within the mitochondrial compartment.”

Increased flux through mitochondrial MEs at least partially sustained AcCoA metabolism in Mpc2KD cells.

“Increased flux through mitochondrial MEs at least partially sustained AcCoA metabolism.”

Mitochondrial pyruvate and mitochondrial alanine aminotransferase (ALT2) play a role in mediating glutamine anaplerosis.

“Notably, alanine levels were significantly decreased in Mpc knockdown cells, highlighting a role for mitochondrial pyruvate and mitochondrial alanine aminotransferase (ALT2) in mediating glutamine anaplerosis which was previously described in cancer cells (Weinberg et al., 2010).”

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Comprehensive 13C MFA integrating parallel tracer experiments using [13C]glucose and [13C]glutamine increases flux resolution throughout central carbon metabolism.

“Comprehensive 13C MFA integrating data from parallel tracer experiments using [13C]glucose and [13C]glutamine was applied to MPC knockdown cells, as this modeling approach has been shown to increase flux resolution throughout central carbon metabolism (Ahn and Antoniewicz, 2013).”

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At high concentrations pyruvate passively enters the matrix, bypassing the MPC.

“At high concentrations pyruvate passively enters the matrix, bypassing the MPC (Bakker and van Dam, 1974; Halestrap, 1975).”

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Mitochondrial substrate utilization can be controlled in terminally differentiated hSKMs by modulating Mpc activity to influence BCAA catabolism.

“These results suggest that mitochondrial substrate utilization can be controlled in terminally differentiated hSKMs by modulating Mpc activity to influence BCAA catabolism.”

Oxidation of mitochondrial substrates other than glutamine was induced upon Mpc depletion.

“Flux from this AcCoA pool into the TCA cycle increased 5-fold in Mpc2KD cells compared to Control cells, suggesting that oxidation of mitochondrial substrates other than glutamine were also induced upon Mpc depletion (Figure 3A and Tables S1-S2).”

These results highlight the flexibility of oxidative mitochondrial metabolism that can be induced by Mpc inhibition such that C2C12 cells meet their energetic demands through glutamine and fatty acid oxidation.

“These results further highlight the flexibility of oxidative mitochondrial metabolism that can be induced by Mpc inhibition such that C2C12 cells meet their energetic demands through glutamine and fatty acid oxidation.”

The lack of change in respiration with UK5099 treatment is presumably due to metabolic plasticity of cells when other pathways (glutamine and fatty acid oxidation) were not inhibited.

“We observed no change in ATP-linked or maximal respiration with UK5099 treatment, presumably due to the metabolic plasticity of cells when other pathways (i.e., glutamine and fatty acid oxidation) were not inhibited (Figures 4D, S2F-S2G for raw values).”

The accumulation of aspartate in Mpc knockdown cells provides evidence of a switch to rely on oxaloacetate and mitochondrial glutamic-oxaloacetic transaminase 2 (GOT2) to facilitate glutamine anaplerosis.

“The accumulation of aspartate in these cells provides evidence of the expected switch to rely on oxaloacetate and mitochondrial glutamic-oxaloacetic transaminase 2 (GOT2) to facilitate this process (Figure S1B).”

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Elevated glutamine anaplerosis and oxidation are likely a consequence of decreased citrate synthase and oxidative IDH flux downstream of PDH while the ETC remained active.

“Elevated glutamine anaplerosis and oxidation are likely a consequence of decreased citrate synthase and oxidative IDH flux downstream of PDH while the ETC remained active (Fan et al., 2013; Le et al., 2012).”

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Knockdown of Mpc2 induced a significant increase in fatty acid oxidation flux in C2C12 myoblasts.

“Increased label incorporation into numerous TCA metabolites downstream of citrate was also detected (Figure 3C), indicating that knockdown of Mpc2 induced a significant increase in fatty acid oxidation flux in C2C12 myoblasts.”

A similar response (as in Mpc knockdown cells) was observed in isolated retina treated with the MPC inhibitor Zaprinast.

“A similar response was observed in isolated retina treated with the MPC inhibitor Zaprinast (Du et al., 2013).”

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Maintenance of the knockdown upon differentiation was confirmed at the protein and mRNA levels.

“Maintenance of the knockdown upon differentiation was confirmed at the protein and mRNA levels (Figures 5A-5B).”

The metabolic response to UK5099 treatment is distinct from that occurring in response to Complex I inhibition.

“As observed in the comparison to hypoxia (Figure 2L), the metabolic response to UK5099 treatment is distinct to that occurring in response to Complex I inhibition.”

An acceptable fit for Mpc2 knockdown cells could only be obtained when an exogenous source of AcCoA was included in the model.

“Intriguingly, an acceptable fit for Mpc2 knockdown cells could only be obtained when an exogenous source of AcCoA was included in the model.”

Inhibition of mitochondrial pyruvate uptake can rewire cellular metabolism to boost fatty acid oxidation.

“Our model, though, suggests that inhibition of mitochondrial pyruvate uptake can rewire cellular metabolism to boost fatty acid oxidation, which may provide an approach to further studies of the interplay between carbohydrate and fat metabolism.”

MPC1 and MPC2 are components of the mitochondrial pyruvate transporter.

“Our analyses also more explicitly delineate the roles of MPC1 and MPC2 as components of the mitochondrial pyruvate transporter.”

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MPC inhibition elicits a distinct metabolic phenotype compared to hypoxia or ETC inhibition.

“These results also demonstrate that MPC inhibition elicits a distinct metabolic phenotype compared to hypoxia or ETC inhibition.”

No evidence of glutaminolysis through malic enzyme was observed in Du et al.

“While Du et al. similarly observed an increase in aspartate levels, no evidence of glutaminolysis through malic enzyme was observed (Du et al., 2013), potentially due to off-target effects on mitochondrial glutaminase (Elhammali et al., 2014).”

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Pharmacological inhibition of the MPC could stimulate catabolic metabolism in muscle or liver.

“In this manner, pharmacological inhibition of the MPC could stimulate catabolic metabolism in muscle or liver.”

The increased flux through malic enzyme could provide additional reducing equivalents [NAD(P)H] within the matrix.

“Citrate mass isotopomer labeling and flux estimations also highlighted a significant increase in flux through malic enzyme upon Mpc knockdown, which could provide additional reducing equivalents [NAD(P)H] within the matrix.”

Mpc inhibition may potentiate oxidation of BCAAs or other amino acids that accumulate in the context of insulin resistance.

“Mpc inhibition may potentiate oxidation of BCAAs or other amino acids that accumulate in the context of insulin resistance (Adams, 2011; Newgard et al., 2009; Wang et al., 2011).”

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Bricker et al. and Herzig et al. both observed growth defects in medium lacking leucine or valine in their characterizations of yeast with Mpc deletions.

“Consistent with this change, Bricker et al. (Bricker et al., 2012) and Herzig et al. (Herzig et al., 2012) both observed growth defects in medium lacking leucine or valine in their characterizations of yeast with Mpc deletions.”

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β-oxidation was presumably stimulated, in part, due to decreases in citrate and the downstream lipogenic intermediate malonyl-CoA, which inhibits CPT1.

“Here β-oxidation was presumably stimulated, in part, due to decreases in citrate and the downstream lipogenic intermediate malonyl-CoA, which inhibits CPT1 (McGarry et al., 1983).”

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These results are consistent with the severe growth defect previously observed in yeast with deletions in both mpc1 and mitochondrial malic enzyme growing on glucose.

“These results are consistent with the severe growth defect previously observed in yeast with deletions in both mpc1 and mitochondrial malic enzyme growing on glucose (Bricker et al., 2012), highlighting the remarkably conserved nature of metabolism across species.”

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