Lactate activates the mitochondrial electron transport chain independently of its metabolism

Abstract

Lactate has long been considered a cellular waste product. However, we found that as extracellular lactate accumulates, it also enters the mitochondrial matrix and stimulates mitochondrial electron transport chain (ETC) activity. The resulting increase in mitochondrial ATP synthesis suppresses glycolysis and increases the utilization of pyruvate and/or alternative respiratory substrates. The ability of lactate to increase oxidative phosphorylation does not depend on its metabolism. Both L- and D-lactate are effective at enhancing ETC activity and suppressing glycolysis. Furthermore, the selective induction of mitochondrial oxidative phosphorylation by unmetabolized D-lactate reversibly suppressed aerobic glycolysis in both cancer cell lines and proliferating primary cells in an ATP-dependent manner and enabled cell growth on respiratory-dependent bioenergetic substrates. In primary T cells, D-lactate enhanced cell proliferation and effector function. Together, these findings demonstrate that lactate is a critical regulator of the ability of mitochondrial oxidative phosphorylation to suppress glucose fermentation.

Keywords: TCA cycle; electron transport chain; glycolysis; lactate; mitochondria; oxidative phosphorylation.

Figure 1.. Lactate suppresses glycolysis by increasing oxidative phosphorylation.

(A) Proliferation of HepG2 cells cultured DMEM with the indicated glucose and lactate concentrations measured as cell number fold change relative to day 0. Gluc, glucose. (B) Glucose consumption of HepG2 cells cultured in DMEM with 1mM glucose with or without the addition of 20mM L-lactate. (C) Schematic of cytosolic LDH catalyzed oxidation of L-lactate and L-alpha-hydroxybutyrate to their respective alpha-ketoacids. (D) Glucose consumption of HepG2 cells cultured in medium with 5mM glucose with the addition of NaCl, L-lactate, or L-AHB at 20mM each. (E) Glycolytic proton efflux rate (GlycoPER) as measured using the Seahorse Bioanalyzer of HepG2 cells cultured in medium containing 5mM glucose with the addition of NaCl, L-lactate, or L-AHB at 20mM each. (F) Glucose consumption of HepG2 cells containing a doxycycline (Dox) inducible LbNOX vector following the indicated treatments with or without the addition of 20mM L-lactate in medium containing 5mM glucose. (G) Oxygen consumption rate (OCR) of HepG2 cells measured using Seahorse Bioanalyzer. Metabolite arrow indicates injection of either 20mM NaCl (control) or 20mM L-lactate. Oligo, oligomycin; Rot/AA, rotenone/antimycin A. (H) Glucose consumption of HepG2 cells cultured in 2mM glucose following the indicated treatment with or without the addition of 10mM L-lactate. Untreated (−) or control conditions in this and subsequent figures indicate equimolar NaCl. All error bars represent mean +/± SD with a minimum n of 3. Statistical analysis in (B), (F), and (H) was performed using two-sided Student’s t-test, in (D) and (E) was performed using one way ANOVA. **** p < 0.0001, * p < 0.05, and ns nonsignificant. See also Figure S1.

Figure 2.. Both stereoisomers of lactate increase oxidative phosphorylation and lactate can suppress glycolysis independent of its metabolism

(A) Schematic of [U-¹³C] L- or D-lactate tracing to pyruvate through expected enzymatic activities of LDHA/B or LDHD. (B) Percent labeling of pyruvate M+3 or citrate M+2 in HepG2 cells following incubation with 10mM [U-¹³C] L- or D-lactate for 8 hours. (C) OCR of HepG2 cells measured using Seahorse Bioanalyzer. Metabolite arrow indicates injection of NaCl (control), L- or D-lactate at 20mM each. (D) Proliferation of HepG2 cells cultured in 1mM glucose with or without the addition of 20mM D-lactate measured by cell number fold change relative to day 0. (E) Glucose consumption of HepG2 cells cultured in medium with 5mM glucose with or without the addition of 20mM D-lactate. (F) OCR and ECAR of HepG2 cells measured using Seahorse Bioanalyzer. Metabolite arrow indicates injection of NaCl (control) or D-lactate at 20mM each. All error bars represent mean +/± SD with a minimum n of 3. Statistical analysis in (D) and (E) was performed using two-sided Student’s t-test. ** p < 0.01, **** p < 0.0001. See also Figure S2.

Figure 3.. Lactate stimulation of mitochondrial respiration increases use of pyruvate as a TCA substrate.

(A) Schematic of simultaneous [U-¹³C] glucose and [3-¹³C] lactate tracing into the TCA. (B and C) Percent labeling of pyruvate and citrate from 10mM [U-¹³C] glucose with indicated concentrations of [3-¹³C] lactate in HepG2 and HEK293 cells. (D) HepG2 cells were treated for the indicated time with 20mM L-lactate or 5mM DCA followed by immunoblotting analysis with the indicated antibodies. (E) HepG2, (F) HEK293, and (G) 143B cells were treated with indicated L-lactate concentrations followed by immunoblotting analysis with the indicated antibodies. (H) HepG2 cells were treated as indicated (20mM NaCl, L- or D-lactate, or 5mM DCA) followed by immunoblotting analysis. (I) Drosophila S2 cells were treated as indicated (20mM NaCl, 5mM DCA, 2mM pyruvate, and 20mM D- or L-lactate) followed by immunoblotting analysis. (J) HepG2 and (K) HEK293 cells cultured in medium containing 25mM or 2.5mM glucose were incubated with 20mM NaCl, L- or D-lactate for 1 hour before total citrate abundance was analyzed using GC-MS. (L) HepG2 and (M) HEK293 cells cultured in 25mM glucose and 2.5mM [U-¹³C] L-lactate were treated with 20mM NaCl, 5mM DCA, or 20mM D-lactate and the percent labeling of pyruvate and citrate from [U-¹³C] L-lactate was analyzed over the time course using GC-MS. All error bars represent mean +/± SD with a minimum n of 3. Statistical analysis in (J) and (K) was performed using one way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001. See also Figure S3.

Figure 4.. Lactate activates the PDH complex in isolated mitochondria in a dose dependent manner and directly enters the mitochondria matrix independent of mitochondrial pyruvate carrier.

(A) Schematic of subcellular fractionation and cell free assay using purified mitochondria. (B) Mitochondria purified from 293T cells were incubated in assay buffer with the indicated treatments (5mM NaCl, DCA, L-lactate, pyruvate, 4mM ATP or ADP) for 30 mins at 30°C, followed by immunoblotting with the indicated antibodies. (C) Purified mitochondria were incubated with indicated concentration of L- or D-lactate (mM), 5mM DCA, or 5mM calcium (Ca, phosphatase activator) followed by immunoblotting analysis. (D) Schematic of [U-¹³C] pyruvate or [U-¹³C] lactate tracing in purified mitochondria. (E and F) Mitochondria purified from 293T cells expressing empty vector or mitochondrial-matrix targeted (Mito-) wildtype or mutant (H193A) LDHA were incubated with or without substrate (2mM [U-¹³C] lactate or 2mM [U-¹³C] pyruvate) in the presence or absence of the MPC blocker UK5099 (10µM), followed by GC-MS analysis of total citrate and α-ketoglutarate M+2 abundance in the reaction mixture. (G) 293T cells expressing mitochondrial-matrix targeted (Mito-) LDHA WT or H193A with or without MPC1 deletion were cultured in media containing 10mM [U¹³C] L-lactate or 2mM [U¹³C] pyruvate followed by GC-MS analysis of citrate percentage labeling. All error bars represent mean ± SD with a minimum n of 3. See also Figure S4.

Figure 5.. Lactate activates the electron transport chain independent of mitochondrial pyruvate entry.

(A) Purified mitochondria were subjected to rapid freeze and thaw or left on ice (untreated), followed by incubation at 30°C for 30 mins in assay buffer with the indicated treatments at 5mM with subsequent immunoblotting analysis. Ca, calcium; Phos-I, phosphatase inhibitor at 1x. (B) Purified mitochondria were resuspended in assay buffer with or without the indicated detergent, followed by cell-free mitochondria assay as described in (A) with the indicated treatments. Phos-I was at 1x, L-lactate was used at 5mM and 15mM, and all others at 5mM. (C) Following overnight culture at the indicated oxygen level, HepG2 cells were treated with 5mM DCA, 15mM or 30mM L-lactate, or 2mM pyruvate for 30 mins at the indicated oxygen level followed by immunoblotting. (D) Following overnight culture at the indicated oxygen level or in 100µM CoCl2, HepG2 cells were treated with 15mM or 30mM L-lactate or 5mM DCA for 30 mins followed by immunoblotting. (E) 143B Rho0 or matched WT control cells were treated with 20mM L-/D-lactate or 5mM DCA for 30 mins followed by immunoblotting. (F and G) Purified mitochondria from 293T or 293T sgDLAT (PDH E2) cells were incubated in assay buffer containing 10mM NaCl (control), 10mM L-/D-lactate, 1µM rotenone, or 1µM FCCP for 30 mins at 30°C, followed by measuring the total NAD⁺ and NADH levels in the reaction using a modified enzyme cycling assay. (H) OCR of HepG2 cells containing the indicated sgRNA measured using Seahorse Bioanalyzer. Metabolite arrow indicates injection of either 20mM NaCl (control), L- or D-lactate. All untreated or control conditions indicate NaCl equimolar to that of the highest lactate concentration. All error bars represent mean ± SD with a minimum n of 3. See also Figure S5.

Figure 6.. D-lactate enhances respiration dependent cell proliferation.

(A to F) 143B and MEF cells were cultured in glucose-deficient DMEM supplemented with galactose (A and D) or complete DMEM containing the indicated rotenone (B and E) or antimycin A (C and F) concentrations, along with the addition of 20mM NaCl or D-lactate. Proliferation was measured by cell number fold change at day 5 relative to day 0. (G) Proliferation of 143B cytochrome-B cybrids (143B CytB) cultured in complete DMEM with uridine and supplemented with NaCl, L-lactate, or D-lactate (20mM each) measured as cell number fold change at day 5 relative to day 0. (H) Proliferation of 143B CytB or 143B Rho0 cells cultured in complete DMEM with uridine and the indicated treatments was measured as cell number fold change at day 6 relative to day 0. (I) Proliferation of 143B CytB cells cultured in complete DMEM with the indicated treatment conditions in the absence of uridine. All error bars represent mean +/± SD with a minimum n of 3. See also Figure S6.

Figure 7.. D-lactate enhances primary T-cell proliferation and effector function.

(A) Mitochondrial OCR of primary murine CD8 T cells in RPMI following NaCl (20mM) or D-lactate (20mM) treatment as measured using Seahorse Bioanalyzer. (B) Glucose consumption of primary murine CD8 T cells cultured in RPMI following NaCl (20mM) or D-lactate (20mM) treatment. (C) Relative cell number of primary murine CD8 T cells cultured in 1mM glucose following NaCl (20mM) or D-lactate (20mM) treatment. (D) IFN-γ production in CD8 T cells cultured in 1mM glucose following NaCl (20mM) or D-lactate (20mM) treatment. (E) Puromycin incorporation in CD8 T cells as determined by flow cytometry following NaCl (20mM) or D-lactate (20mM) treatment. All error bars represent mean +/± SD with a minimum n of 3. Statistical analysis in (B), (C) and (E) was performed using two-sided Student’s t-test. **p < 0.01, ***p < 0.001.