Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase

<div><p>Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP⁺) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC₅₀ 3.93 [3.70–4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP⁺ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP⁺ derivatives. Compounds based on or similar to TPP⁺ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes.</p></div

TPMP inhibits mitochondrial respiration and increases glycolytic activity in intact C2C12 cells.

<p><b>A.</b> Metabolic analysis of cellular respiration in intact C2C12 myoblasts. Cells were treated with different TPMP concentrations or vehicle (deionized H₂O), followed by a sequential injection of 1 μM oligomycin (O), 1 μM FCCP (F), then 1 μM rotenone and antimycin A (R+A) mixture. The TPMP treated cells showed a decrease in oxygen consumption rate (OCR) following the TPMP addition. The inhibitory effect following TPMP treatment is demonstrated by a gradual dose-dependent decrease in cellular OCR. Data is expressed as the percentage of basal OCR (OCR%) and is presented as means ±95% CI, n = 4, measured in triplicate. <b>B.</b> A corresponding increase in ECAR following TPMP addition. Data is expressed as the percentage of basal ECAR (ECAR%) and is presented as means ±95% CI, n = 4, measured in triplicate. <b>C.</b> IC₅₀ shift showing the time dependent inhibition after 20, 40, and 60 minutes of TPMP treatment. <b>D.</b> IC₅₀ for mitochondrial ATP driven respiration represents difference between respiration after oligomycin addition and respiration in the steady state, which includes proton leak respiration. <b>E.</b> An increase in IC₅₀ of uncoupled respiration following the induced collapse of membrane potential by FCCP and the leak of the accumulated TPMP inside mitochondria. In C, D & E mitochondrial OCR was calculated by subtracting non-mitochondrial respiration after rotenone-antimycin A treatment, then the values were plotted against logarithm values of TPMP in micromolar concentration (log TPMP μM vs. normalized response %). IC₅₀ was then estimated by non-linear regression analysis. <b>F.</b> Detection of changes in Δ<i>ψ</i> induced by 10 μM TPMP and mitochondrial inhibitors expressed as changes in TMRM fluorescence (ex = 520 nm and em = 580 nm). We perturbed mitochondrial respiration by different inhibitors to induce different states of membrane polarization. The groups included an untreated control (polarized), 1 μM oligomycin treated (hyperpolarized), 0.5 μM FCCP treated (depolarized) and 1 μM rotenone treated (decreased polarization). Data is expressed as TMRM fluorescence (a.u) means ±95% CI, n = 4, in which each group included 10 wells. *, p = 0.0013; #, p = 0.0094; **, p<0.0001; ##, p = 0.0076; ns, not significant.</p

Respiration in permeabilized C2C12 cells in the presence of different metabolic substrates.

<p>Respiration was induced by the addition of ADP in the presence of the relevant substrate. Cells were treated with TPMP 10 μM or vehicle (deionized H₂O), followed by 1 μM FCCP, then 1 μM AA to block the flow of electrons in the respiratory chain. <b>A.</b> Respiration on 5mM pyruvate/ 2.5 mM malate. <b>B.</b> Respiration on 5 mM glutamate / 2.5 mM malate. <b>C.</b> Respiration on 10 mM succinate. <b>D & E.</b> The acidification rate (PPR) increased following TPMP treatment in pyruvate/malate and glutamate/malate cases. <b>F.</b> In the presence of succinate, the increase in PPR occurred after AA addition. Data is expressed as the percentage of basal OCR (OCR%) and is presented as means ±95% CI, n = 4, measured in triplicate. AA, antimycin A; OCR, oxygen consumption rate; PPR, proton production rate.</p

A schematic illustrates TPMP distribution.

<p>Due to the membrane potential, lipophilic cations tend to achieve Nernstian equilibrium across biological membranes.</p

A schematic presentation of Krebs cycle showing the active enzymes when different mitochondrial substrates are used.

<p>NADH produced is re-oxidized by the respiratory complex I. The role of malate in pyr/mal respiration is to provide oxaloacetate that is further metabolized to citrate in the presence of acetyl-CoA. In glu/mal respiration, malate takes part in the malate aspartate shuttle. PDHC, pyruvate dehydrogenase complex; A-CoA, acetyl-CoA; CS, citrate synthase; C, citrate; ACN, aconitase; IC, isocitrate; IDH, isocitrate dehydrogenase; GDH, glutamate dehydrogenase; 2-OG, 2-oxoglutarate; OGDHC, 2-oxoglutarate dehydrogenase complex; S-CoA, succinyl-CoA; SCS, succinyl-CoA synthase; S, succinate; SDH, succinate dehydrogenase; F, fumarate; FH, fumarate hydratase; M, malate; MDH, malate dehydrogenase; OA, oxaloacetate; Q, ubiquinone; QH₂, ubiquinol.</p

Inhibition of OGDHC by TPMP.

<p><b>A.</b> The enzymatic activities of Krebs cycle and electron transport chain were not affected by the presence of 1 mM TPMP, except the OGDHC, which was significantly reduced. Data is presented as means ±95% CI, n = 3. * indicates significant p<0.05. Respiratory chain complexes and Krebs cycle enzymes were assessed in rat skeletal muscle homogenate enriched in mitochondrial fraction that was exposed to 3 freeze-thaw cycles, with triton X-100 0.1% included in the reaction buffer to ensure complete permeabilization of any remaining intact mitochondria. Pyruvate dehydrogenase complex was obtained purified from porcine heart. MDH, malate dehydrogenase; CS, citrate synthase; IDH, isocitrate dehydrogenase; OGDHC, 2-oxoglutarate dehydrogenase complex; PDHC, pyruvate dehydrogenase complex; GDH, glutamate dehydrogenase. <b>B.</b> IC₅₀ of TPMP was found to be 3.93 [3.70–4.17] mM. Data is presented as means ±95% CI, n = 3. <b>C.</b> Longer chain alkyl-TPP⁺ compounds have higher inhibitory effect. OGDHC activity in the presence of 1 mM concentration of the more lipophilic TPP⁺ moieties in the assay mixture. The rate of inhibition was proportional to the increase in the length of the alkyl side chain. OGDHC activity was measured as in A. Data is presented as means ±95% CI, n = 3. TPMP, methyltriphenylphosphonium; Propyl, propyltriphenylphosphonium; Pentyl, pentyltriphenylphosphonium. <b>D. & E.</b> Comparison of the metabolite level in the vehicle treated control (deionized H₂O) and TPMP treated cells in a cellular suspension (≈4 million cells in 10 ml DMEM). <b>D.</b> The concentration of lactate in the supernatant increased in the TPMP treated group after 1 hour of incubation. Data is presented as means ±95% CI, n = 4. *, p = 0.0023. <b>E.</b> The level of 2-oxoglutarate increased in the cellular pellet in the TPMP treated cells after inhibition of OGDHC complex. Data is presented as means ±95% CI, n = 4. *, p = 0.0012.</p

The Effect of Pericellular Oxygen Levels on Proteomic Profile and Lipogenesis in 3T3-L1 Differentiated Preadipocytes Cultured on Gas-Permeable Cultureware

<div><p>Pericellular oxygen concentration represents an important factor in the regulation of cell functions, including cell differentiation, growth and mitochondrial energy metabolism. Hypoxia in adipose tissue has been associated with altered adipokine secretion profile and suggested as a possible factor in the development of type 2 diabetes. <i>In vitro</i> experiments provide an indispensable tool in metabolic research, however, physical laws of gas diffusion make prolonged exposure of adherent cells to desired pericellular O₂ concentrations questionable. The aim of this study was to investigate the direct effect of various O₂ levels (1%, 4% and 20% O₂) on the proteomic profile and triglyceride accumulation in 3T3-L1 differentiated preadipocytes using gas-permeable cultureware. Following differentiation of cells under desired pericellular O₂ concentrations, cell lysates were subjected to two-dimensional gel electrophoresis and protein visualization using Coomassie blue staining. Spots showing differential expression under hypoxia were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. All identified proteins were subjected to pathway analysis. We observed that protein expression of 26 spots was reproducibly affected by 4% and 1% O₂ (17 upregulated and 9 downregulated). Pathway analysis showed that mitochondrial energy metabolism and triglyceride synthesis were significantly upregulated by hypoxia. In conclusion, this study demonstrated the direct effects of pericellular O₂ levels on adipocyte energy metabolism and triglyceride synthesis, probably mediated through the reversed tricarboxylic acid cycle flux.</p></div

Proteins downregulated in 1% O₂ and 4% O₂ compared to control conditions (20% O₂).

<p>Proteins downregulated in 1% O₂ and 4% O₂ compared to control conditions (20% O₂).</p