UK5099 inhibits macrophage activation independent of mitochondrial pyruvate carrier mediated metabolism

Glycolysis is essential for the classical activation of macrophages (M1), but how glycolytic pathway metabolites engage in this process remains to be elucidated. Glycolysis culminates in the production of pyruvate, which can be transported into the mitochondria by the mitochondrial pyruvate carrier (MPC) followed by conversion to citrate and utilization in the TCA cycle. Alternatively, pyruvate can be metabolized to lactate under aerobic conditions, which had been considered to be the dominant route in the setting of classical macrophage activation. However, based on studies that used UK5099 as a MPC inhibitor and showed reduction in key inammatory cytokines, the mitochondrial route has been considered to be of signicance for M1 activation as well. Herein, using a genetic depletion model, we found that MPC is dispensable for metabolic reprogramming and the activation of M1. While UK5099 reaches maximal MPC inhibitory capacity at approximately 2–5µM, higher concentrations are required to inhibit inammatory cytokine production in M1 and this is independent of MPC expression. Apart from MPC inhibition, UK5099 at high doses impairs glutamate oxidation, mitochondrial membrane potential and HIF-1α stabilization. Taken together, UK5099 inhibits inammatory responses in M1 macrophages due to effects other than MPC inhibition. Samples were vortexed for 20 seconds and transferred to 5 mm NMR tubes. NMR spectra were acquired on a Bruker 500 MHz Avance III HD spectrometer equipped with a BBO cryoprobe and SampleCase auto sampler (Bruker Biospin, Rheinstetten, Germany). 1 H-NMR spectra were recorded using 1D noesy pulse sequence with presaturation (noesygppr1d), with 90 degree pulse (~13 µs), 4.68 seconds acquisition time, and 4 seconds relaxation delay. Spectra were phase and baseline corrected using the Topspin 3.5 software. Metabolites were identied and quantied using the software program Chenomx NMR Suite 8.2, by tting the spectral lines of library compounds into the recorded NMR spectrum of the cell extracts. quantication peak area TSP-d and metabolite concentrations in


Introduction
A growing body of knowledge demonstrates that the switch from oxidative phosphorylation to glycolysis plays a critical role in the in ammatory response of macrophages when stimulated with agents such as lipopolysaccharide (LPS) (classical activation) [1,2,3,4]. Pyruvate, the last product of glycolysis, can be transferred into the mitochondria to fuel the tricarboxylic acid (TCA) cycle as citrate or it can be metabolized into lactate. The latter route, known as "aerobic glycolysis" or the "Warburg effect", is considered to be the main nal pathway in classically activated macrophages, allowing them to use (repurpose) the mitochondria for reactive oxygen species (ROS) generation rather than ATP production [5][6][7]. Consequently, ROS production stabilizes hypoxia induced factor 1α (HIF-1α) and promotes proin ammatory cytokines production [5][6][7]. The role of the alternative, mitochondrial route of pyruvate in this setting is less clear. The transport of pyruvate across the mitochondrial membrane is an active process, enabled by the mitochondrial pyruvate carrier (MPC) complex, which consists of two subunits: MPC1 and MPC2 [8,9]. Interestingly, some studies found MPC-mediated metabolism to also be important for the activation of LPS-stimulated macrophages [10,11,12,13]. In this setting, the theory has been that pyruvate transportation by MPC is required for acetyl-CoA production, histone acetylation, and epigenetic changes, which then facilitate in ammatory gene expression [10,11]. However, these ndings and interpretations are established based on pharmacological inhibition of MPC using α-cyano-β-(1phenylindol-3-yl)-acrylate (UK5099) as such an inhibitor [10,12,13]. Therefore, closer examination of the MPC inhibitor UK5099 is warranted to clearly interpret the role of MPC in macrophage activation.
In this study, we evaluated the role of MPC-mediated metabolism in LPS-activated macrophages using a murine Mpc conditional knockout model. Surprisingly, we found that Mpc depletion has no impact on in ammatory cytokines production in macrophages. Moreover, UK5099 reduces cytokine production of bone marrow derived macrophages (BMDMs) from either wild-type (WT) mice or mice that lack MPC expression in myeloid cells. RNA sequencing analysis indicated that excessive concentrations of UK5099 have a much broader impact on gene expression than Mpc depletion in LPS-stimulated macrophages. UK5099 at high doses could effectively suppress the in ammatory response, while a low dose of 2-10 μM failed to do so despite being equally potent to reduce the transport of glucose-derived pyruvate into the mitochondria. Importantly, UK5099 not only inhibits MPC but also glutamate oxidation and HIF-1α stabilization. Based on these results, we conclude that UK5099 possesses off-target effects at high concentrations, suggesting that experimental investigations utilizing UK5099 should be interpreted with caution.

Results
Generating mice with myeloid cell-speci c deletion of Mpc To test if MPC-mediated transportation of glycolysis-derived pyruvate is required for the activation of macrophages by LPS, we rst generated Mpc1oxed mice by CRISPR/dCas9/gRNA (Mpc1 / ) ( Figure   1a). Breeding with Lyz2-Cre mice yielded mice with myeloid cell-speci c deletion of Mpc1 (Mpc1 -/-). As shown in Figure 1b, Mpc1 mRNA level in bone marrow derived macrophages (BMDMs) isolated from Mpc1 -/mice was dramatically reduced compared with those from Mpc1 / mice, while Mpc2 mRNA level remained unchanged (Figure 1c). Neither MPC1 nor MPC2 protein was expressed in Mpc1 -/-BMDMs (Figure 1d and 1e), and both proteins are known to be indispensable to form a stable MPC complex [8,9,14].
Mpc depletion reduces glucose fuel into TCA cycle but has no impact on metabolic reprogramming in LPS-stimulated macrophages MPC-mediated transportation of pyruvate into mitochondria is a critical step for cell metabolism. 13 Cglucose tracing showed that Mpc1 -/-BMDMs had a signi cantly lower ratio of glucose-labeled TCA cycle metabolites compared with Mpc1 / BMDMs (Figure 1f to 1j), indicating effective reduction of glucose ux into the mitochondria. Glycolysis itself was unaffected by Mpc depletion as levels of glucose labeled glycolytic metabolites did not differ between Mpc1 -/and Mpc1 / BMDMs (Figure 1k to 1l). While the upstream TCA cycle metabolites citrate and succinate were reduced (Figure 1m and 1n), downstream metabolites were similar between Mpc1 -/and Mpc1 / BMDMs (Figure 1o to 1p). There was also no difference in the total amounts of intracellular pyruvate and lactate (Figure 1q and 1r). Importantly, Mpc depletion did not affect the metabolic reprogramming of macrophages stimulated by LPS, neither early increase in glycolysis (Figure 2a), nor later decrease in oxidative phosphorylation ( Figure 2b). Likewise, glucose consumption and lactate production signi cantly increased after LPS stimulation to similar levels in Mpc1 -/and Mpc1 / BMDMs (Figure 2c and 2d). However, more pyruvate was secreted into the medium from Mpc1 -/cells (Figure 2e). Mpc depletion also had no impact on cell viability (Figure 2f). The observation that oxidative phosphorylation remained intact after Mpc depletion despite less pyruvate availability in the mitochondria to fuel the TCA cycle points to other compensatory sources. Indeed, Mpc1 -/-BMDMs consumed more glutamine ( Figure 2g) and had higher glutamine labeled TCA cycle metabolites compared with Mpc1 / BMDMs (Figure 2h to 2k), pointing to glutamine as a compensatory source and alternative substrate for oxidative phosphorylation.
Mpc expression is dispensable for proin ammatory cytokines production in LPS-stimulated macrophages We next tested if Mpc depletion in uences the in ammatory response of macrophages stimulated by LPS. Previous studies reported that MPC inhibition by UK5099 reduced the production of proin ammatory cytokines, including TNF-α, IL-6, IL-12 [10, 11, 12, 13]. Genetic Mpc depletion, as in this study, however, did not change the expression of these cytokines (Figure 3a to 3c) or their secretion (Figure 3d to 3f).
Previous studies indicated that the conversion of glycolysis-derived pyruvate to acetyl-CoA supports histone acetylation, which is deemed critical for in ammatory gene expression in M1 macrophages [10].
In the current study Mpc depletion in BMDMs did not decrease histone acetylation ( Figure 3g). Collectively, these data indicated that MPC mediated transportation of glucose-derived pyruvate into the mitochondria is not mandatory for histone acetylation or the activation of macrophages by LPS.
UK5099 suppresses in ammatory responses independent of MPC expression UK5099 has been used as a potent inhibitor of mitochondrial pyruvate carrier and was shown to be effective in reducing the in ammatory response of macrophages [10,11,12,13]. Likewise, in the current study, we found that UK5099 at previously reported doses (100μM) signi cantly reduced both mRNA (Figure 4a to 4c) and protein (Figure 4d to 4f) levels of TNF-α, IL-6 and IL-12 in M1 macrophages without affecting their viability (Figure 4g). This suppressive effect was not only seen in Mpc1 / macrophages but also in macrophages devoid of MPC expression (Figure 4a to 4f), indicating that UK5099 inhibits the in ammatory response of M1 macrophages independent of MPC. RNA sequencing analysis con rmed that Mpc depletion had no impact on proin ammatory cytokine production contrary to UK5099 treatment (Figure 4h-4j). Approximately 1400 genes including TNF-α, IL-6 and IL-12 were changed by UK5099 treatment in LPS stimulated BMDMs, while only 168 genes were altered by Mpc depletion (Figure 4h to 4j). Importantly, these gene expression patterns were similar between Mpc1 / and Mpc1 -/-BMDMs after UK5099 treatment (Figure 4k). GO analysis indicated that UK5099 affects immune responses and cytokines production of LPS stimulated macrophages very broadly ( Figure 4l).

UK5099-suppressed in ammatory cytokines production is concentration dependent
We next inquired about the dose dependency of the effects of UK5099 in M1 macrophages. As shown in Figure 5a, cell viability of LPS stimulated macrophages was similar across the different concentrations of UK5099. On the contrary, there was a clear inhibitory, dose-dependent effect of UK5099 on proin ammatory cytokine production greater than the threshold dose of 25μM (Figure 5b to 5d). This effect was also con rmed by mRNA levels of these proin ammatory cytokines (Figure 5e-5g).
Importantly, no corresponding inhibitory effect on MPC was observed at UK5099 concentrations correlated with reduced proin ammatory cytokine production. As shown in Figure 5h to 5n, UK5099 reduced the ratio of 13 C-glucose labeled TCA cycle metabolites starting at concentrations as low as 1μM.
This inhibitory effect reached a plateau starting at 5μM without any further decrease in 13 C-labeling ratio at any higher concentrations. Moreover, the dose-dependent inhibition of cytokine production by UK5099 did not match the total amounts of TCA metabolites (Figure 5o to 5r). Collectively, UK5099 effectively inhibits MPC at concentrations under 5μM, but its suppressive effect on the in ammatory response of M1 macrophages requires high concentrations and is unrelated to the suppressive effect on MPC.

UK5099 suppresses oxidative phosphorylation (OXPHOS) and mitochondrial membrane potential
Previous studies concluded that UK5099 reduced the entry of pyruvate into the TCA cycle and in turn decreased the incorporation of glucose-derived carbons into histone acetylation [10]. In this study, however, we found that UK5099 treatment resulted in increased global histone acetylation (Figure 6a and 6b) irrespective of impaired pyruvate transport into the mitochondria (Figure 5h to 5n). This nding then generated the question of how UK5099 inhibits the in ammatory response of macrophages. Seahorse experiments showed that low concentrations of UK5099 slightly reduced basal OCR, while high concentrations slightly increased it (Figure 6c and 6d). The intracellular ATP levels were not affected by different concentrations of UK5099 (Figure 6e and 6f). Importantly, high dose UK5099 reduced FCCPstimulated OCR in a dose-dependent manner (Figure 6c and 6d). This trend matched the dose response of UK5099 in inhibiting in ammatory cytokine production. Similar effects were observed in Mpc1 / BMDMs as UK5099 below 10μM slightly reduced FCCP-stimulated OCR but 100μM UK5099 signi cantly decreased it (Figure 6g and 6h). Critically, high (but not low) dose UK5099 also reduced FCCP-stimulated OCR in Mpc1 -/-BMDMs, further indicating that high concentrations of UK5099 in uence cellular metabolism independent of MPC expression (Figure 6i and 6j).
UK5099 in uences FCCP-stimulated maximum OCR more than basal OCR and ATP linked OCR, a portrait that is in line with OXPHOS inhibitors [15]. To determine the underlining mechanism(s) of respiratory inhibition by high concentrations of UK5099, we permeabilized BMDMs and provided cells with different oxidizable metabolites [15,16,17,18]. As shown in Figure 6k to 6m, both Mpc depletion and UK5099 treatment effectively inhibited respiration driven by pyruvate. Of note, 2μM UK5099 reduced respiration to the similar levels to that of 100μM UK5099 and Mpc depletion, indicating that low concentrations of UK5099 are also able to effectively block the entry of pyruvate into TCA cycle. Therefore, the suppressive effect of 100 μM UK5099 on FCCP-stimulated respiration cannot be attributed to the blocked entry of pyruvate into the TCA cycle and OXPHOS, but instead may be due to inhibited oxidation of other offtarget substrates.
We then evaluated the impact of UK5099 on respiration driven by glutamate, whose transportation into mitochondrial and subsequent metabolism is independent of MPC [15, 16, 17, 18]. As expected, Mpc depletion had no impact on glutamate driven respiration (Figure 6n to 6p). However, 100μM UK5099, but not 2 and 10μM UK5099, signi cantly reduced glutamate driven respiration in both Mpc1 / and Mpc1 -/-BMDMs, suggesting that high concentrations of UK5099 not only inhibit MPC but also suppress the utilization of glutamate. This effect is due to the inhibition of later steps of glutamate oxidation rather than less glutamate/glutamine fuel into TCA cycle, as high concentrations of UK5099 did not reduce 13 Cglutamine labeled TCA cycle metabolites (Figure 6q to 6t) and glutamine consumption (Figure 6u).
Since dysfunctional cellular respiration is known to be associated with impaired mitochondrial membrane potential [19, 20, 21, 22], we next tested the mitochondrial membrane potential of BMDMs after treatment with different concentrations of UK5099. As shown in Figure 6v, 100μM UK5099, but not 2 and 10μM UK5099, signi cantly reduced mitochondrial membrane potential.

Discussion
Metabolic reprogramming of immune cells has been investigated intensively in recent years, but better understanding of how metabolism precisely controls immune reponses of these cells is still needed [1][2][3][4][5].
In this study, we demonstrated that MPC mediated metabolism is not required for M1 macrophage activation and targeting MPC alone is unable to reduce macrophage mediated in ammation. We also found that UK5099 not only inhibits MPC but also suppresses OXPHOS, revealing unreported off-target effects of this wildly used inhibitor in the eld of metabolism.

MPC-mediated metabolism is dispensable for the activation of LPS stimulated macrophages
After stimulation by LPS for 18 to 24 hours, one key metabolic signature of macrophages is the reduced oxygen consumption, increased mitochondrial membrane potential and reactive oxygen production generated by the electron transport chain [5,7]. This process promotes HIF-1α stabilization to facilitate pro-in ammatory cytokines production [5,7]. Interestingly, recent studies found that short term stimulation of BMDMs by LPS increases maximal respiratory capacity while maintaining an intact basal respiration, and that increased respiration is required for proin ammatory cytokine production [10, 11]. How glycolytic metabolites participate in this metabolic rewiring of LPS stimulated macrophages, however, remains unclear.
MPC-mediated metabolism is at the intersection of glycolysis, the TCA cycle and OXPHOS. Previous studies demonstrated that pharmacological inhibition of MPC by UK5099 attenuated glycolytic fueling of the mitochondria and the in ammatory response in LPS stimulated macrophages [10, 11, 12, 13]. Herein, however, we found that genetic depletion of Mpc has no effect on metabolic reprogramming and proin ammatory cytokine production in LPS stimulated macrophages. Although Mpc depletion substantially reduced the transportation of glycolysis-derived pyruvate into the TCA cycle, TCA cycle metabolites can be compensated for by other sources, such as glutamine, and therefore elevated maximal respiratory capacity is maintained to promote in ammatory responses. Indeed, previous studies indicated that the main source of accumulated TCA cycle metabolites in LPS stimulated macrophages was glutamine rather than glucose [6]. Collectively, our study supports the notion that the metabolic reprogramming of mitochondria is exible and does not depend on MPC in LPS-activated macrophages.

Histone acetylation and macrophage activation
The role of histone acetylation in macrophage activation is controversial. Prior studies observed that LPS stimulation increases the incorporation of glucose-derived carbons into histone acetylation and that pharmacological inhibition of glycolysis by 2-DG or MPC by UK5099 reduces pro-in ammatory cytokines production. These observations led to the conclusion that the transportation of glycolysis-derived pyruvate into the mitochondria is essential for acetyl-CoA production, histone acetylation, and the activation of LPS stimulated macrophages [10, 11]. However, whether these inhibitory effects impact gobal levels of (rather than glucose incorporated) histone acetylation is unknown. Moreover, recent studies showed that genetic knockdown or depletion of ATP citrate lyase, which catalyzes citrate into acetyl-CoA in the cytoplasm, even increases cytokine production in LPS stimulated macrophages [25,26].
Our study surprisingly found that both Mpc depletion and UK5099 treatment increased histone acetylation, even though intercellular citrate levels were reduced in both conditions. There are two possible reasons for these data. First, cytosolic acetyl-CoA rather than mitochondrial acetyl-CoA is the substrate for histone acetylation. Although Mpc  , leding to the conclusion that mitochondrial entry of pyruvate produced from enhanced glycolysis is critical for the activation of macrophages by LPS. In the current study, by using mice with genetic depletion of Mpc, we found that the inhibition of in ammatory cytokine production in LPS-stimulated macrophages by UK5099 is independent of MPC but shows concentration dependency. Selective MPC inhibitory effects of UK5099 are seen only in a narrow, low-dose range (<10μM). Thus, conclusions drawn from studies on mitochondrial pyruvate transfer using higher doses of UK5099 than this should be evaluated carefully.

Excessive UK5099 impairs OXPHOS and HIF-1α stabilization
Although UK5099 suppresses the in ammatory responses of LPS stimulated macrophages due to its offtarget effects rather than MPC inhibition, elucidating its true targets and mechanism of action is important to identify new compounds to treat in ammatory diseases. UK5099 at high concentrations (e.g 100 μM) effectively inhibits the production of key proin ammatory cytokines, including TNF-α, IL-6 and IL-12, suggesting that appropriate structural modi cation of this drug could be a promising strategy to curb excessive in ammatory responses. Of note, we found that UK5099 at high concentrations not only reduces the transportation of glucose-derived pyruvate into the TCA cycle but also suppresses the oxidation of other substrates, such as glutamate. As the transportation and metabolism of glutamate is independent of MPC, the inhibition of glutamate oxidation is at least one of the off-target effects of UK5099. On the other hand, it is unknown whether MPC inhibition is required in combination with the off targets of UK5099 to suppress in ammatory responses. Although MPC inhibition alone is unable to reduce proin ammatory cytokines production in LPS stimulated macrophages, it is possible that it further decreases available substrates for OXPHOS when glutamate oxidation is impaired by high concentrations of UK5099. Further studies are required to investigate how macrophages precisely control metabolic reprogramming in mitochondria to ne tune in ammatory responses.
In conclusion, we found that MPC mediated metabolism is dispensable for the activation of LPS stimulated macrophages. UK5099 inhibits the in ammatory response in macrophages, but this is due to its off-target effects rather than MPC inhibition. The mice were bred and maintained under pathogen-free conditions with ad libitum access to food and water. This study was approved by the Tongji University Institutional Animal Use and Care Committee (IACUC).

Bone Marrow Derived Macrophage Isolation
Bone marrow-derived macrophages (BMDMs) were isolated as previously described (34-36). Brie y, mice were individually euthanized by CO 2 inhalation, and both the femur and tibia were isolated and placed in PBS after removing the skin and muscle. Bone marrow cells were then ushed out from the bone, resuspended and grown in RPMI-1640 media (10% heat-inactivated FBS and 1% penicillin and streptomycin) containing 20 ng/ml M-CSF. Half the volume of fresh growth medium was added on day 4. After 7 days in culture, macrophages were harvested and plated for further experimentation.

Macrophage activation
BMDMs were seeded at 1×10 6 /mL for experiments unless stated otherwise. The cells were activated with LPS (100 ng/mL) for the indicated times.  Samples were vortexed for 20 seconds and transferred to 5 mm NMR tubes. NMR spectra were acquired on a Bruker 500 MHz Avance III HD spectrometer equipped with a BBO cryoprobe and SampleCase auto sampler (Bruker Biospin, Rheinstetten, Germany). 1 H-NMR spectra were recorded using 1D noesy pulse sequence with presaturation (noesygppr1d), with 90 degree pulse (~13 µs), 4.68 seconds acquisition time, and 4 seconds relaxation delay. Spectra were phase and baseline corrected using the Topspin 3.5 software. Metabolites were identi ed and quanti ed using the software program Chenomx NMR Suite 8.2, by tting the spectral lines of library compounds into the recorded NMR spectrum of the cell extracts.
The quanti cation was based on peak area of TSP-d 4 signal, and metabolite concentrations were reported as µM in medium.

C 6 -glucose and C 5 -glutamine tracing experiments
Metabolic tracing analysis of U-[ 13 C]-glucose, U-[ 13 C]-glutamine and 2-13 C-pyruvate in BMDMs was determined by GC/MS. Brie y, 6×10 6 cells were incubated in RPMI-1640 medium containing 11.1 mM 13 C 6 -glucose, 2 mM 13 C 5 -glutamine or 1 mM 2-13 C-pyruvate. Cells were treated with or without different concentrations of UK5099 for 1 hour, followed by stimulation with LPS for 4 hours. Samples were collected and processed as described for intracellular metabolite measurement. Metabolites were quanti ed based on total ion count peak area of speci c mass ions. To determine 13 C-labeling, mass information for known fragments of labeled metabolites was retrieved. These fragments contained either the whole or partial carbon skeleton of the metabolite. For each fragment, the retrieved data comprised mass intensities for the lightest isotopomer (without any heavy isotopes, M+0), and isotopomers with increasing unit mass (up to M+6) relative to M0. These mass distributions were normalized by dividing by the sum of M0 to M6, and corrected for the natural abundance of heavy isotopes, using matrix-based probabilistic methods as described (37)  Santa Clara, CA), as described previously (36). Brie y, a phosphate buffer with tetrabutylammonium sulfate and methanol mixture was used as mobile phase at 0.7 ml/min ow rate. Gradient elution was applied and the separation of nucleotides was completed in 10 minutes. The nucleotide levels were normalized by cell number.

Mitochondrial membrane potential measurement
Cells were plated at 0.5×10 6 cells/ml in 12-well plates and treated with different concentrations of UK5099 for 1 hour, followed by stimulation with LPS for 4 hours. After removing culture media, cells were washed and incubated in PBS containing 50nM Mito Tracker Red (Thermo Fisher Scienti c) at 37°C in the dark for 30 min. After 3 washes, cells were then collected in 500 μl PBS by using a cell scraper and transferred to polypropylene FACS tubes. Aqua uorescent reactive dye (Thermo Fisher Scienti c) was applied to excluded dead cells. Cells were then analyzed using a LSRFortessa ow cytometer, and data were analyzed using FlowJo software.

Gene expression analysis by 3' RNA sequencing
Mpc1 / and Mpc1 -/-BMDMs were seeded in 12 well plates (1*10 6 /well) and stimulated with or without LPS for 4 h after pretreatment with UK5099 (100μM) or DMSO for 1 h. After washing with PBS, cells were collected in Trizol (Thermo Fisher Scienti c) and stored in liquid nitrogen. RNA was extracted and a total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Brie y, mRNA was puri ed from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature and the library fragments were puri ed with Cell viability were seeded in 96-well plates and treated with different reagents before stimulation with or without LPS for the indicated times. Each well was added with 50 μl XTT detection solution and absorbance was read at 450 nm after incubation at 37 o C for 3h.

Statistical Analysis
Results were presented as mean ± standard error of the mean (sem). The unpaired Student's t-test was used to test the differences between two groups, based on the assessment of variance of the data. All data were analyzed by GraphPad Prism software (version 7). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Detailed statistical values are provided in the gure legends.

DATA AND CODE AVAILABLE
The raw RNA sequencing data les reported in this study is uploading to NCBI and will be ready for access at any stage during the submission.      Vertical line indicates initial injection of the activator. ***p < 0.001, ****p < 0.0001; ns, no signi cant difference. Data are representative of three independent experiments (n = 8, mean ± SEM). (n) to (p) Mpc / and Mpc-/-BMDMs were stimulated by LPS for 4 hours ± 1 hour pre-treatment with different concentrations of UK5099. The cells were then permeabilized in MAS buffer and provided with glutamate (10mM)/malate (5mM), after which OCR was measured. The arrows indicate injection of the activator. **p < 0.01; ns, no signi cant difference. Data are representative of three independent experiments (n = 8, mean ± SEM).