Additional file 1 of Lactate oxidative phosphorylation by annulus fibrosus cells: evidence for lactate-dependent metabolic symbiosis in intervertebral discs

Additional file 1: Figure S1. Lactate tolerance by AF cells in cell culture. Rabbit AF cells were grown on monolayer culture under physiologic nutrients, e.g. 1% FBS, 1.06 mM glucose and varying exogenously added lactate concentrations (0-20 mM), for 48 hrs. Effects of different lactate concentrations on cell density and morphology (A) and cell viability as determined by CCK8 assay (B). Cell viability data are means ± SEM of three independent experiments (3 rabbits). Figure S2. Lactate import into HepG2 cells. 14C-lactate radioactive tracing to assess lactate import into HepG2 cell cultures grown in 1mM glucose and varying concentrations of 14C-lactate (1, 4mM) for one minute, washed with PBS, lysed and counted in scintillation fluid. Figure S3. Lactate conversion to pyruvate in AF cells in ex vivo disc organ. 13C-lactate tracing to pyruvate conversion in ex vivo rabbit disc organ culture containing 4 mM 13C-lactate in the culture media. Intracellular enrichment of 13C lactate or pyruvate AF cells from AF tissue of the ex vivo disc organ culture is reported as atomic percent excess (APE) of the total amount of lactate or pyruvate, e.g. 10% APE of pyruvate indicates 10% of total pyruvate contains 13C. Percent (%) APE shown. (M+1) indicates that one 13C carbon is present on lactate or pyruvate molecule. Figure S4. Lactate uptake and conversion to TCA intermediates and amino acids in rabbit, human, and rat AF cells. (A) 13C from rabbit AF cells cultured in 4 mM 3-13C-lactate and 1mM glucose was traced to amino acids glutamate, glutamine, and alanine. (B) Preferential lactate uptake and conversion to TCA intermediates by rabbit AF cells. 13C from 13C -Lactate or 13C -Glucose was traced by HRMS to TCA intermediates in rabbit AF cells grown in three different culture media. Note that 13C enrichments of succinate, fumarate, and malate from 1mM 13C -Glucose (black bars) were dramatically reduced in the presence of 4 mM unlabeled lactate (grey bars). Consistent with this result is that 13C enrichments of succinate, fumarate, and malate from 4mM 13C -Lactate were not affected in 1mM unlabeled glucose (white bars), indicating that AF cells preferentially utilize lactate when grown under physiologic nutrient condition of 1mM glucose and 4mM lactate. (C) 13C from human AF cells cultured in 4 mM 3-13C-lactate was traced to the TCA intermediate malate and the amino acid glutamate. (D) Caudal discs of Fischer 344 rats were injected with 3-13C-lactate (see method) and 13C was traced to lactate, malate, and glutamate in the AF tissues 3 days post injection. Intracellular enrichment of 13C on different metabolites in AF cells was measured in APE or atom percent excess. Data are means ± SEM of three independent experiments (three rats) for D, four experiments for C (four human disc specimen), and four experiments for A, B (four rabbits). Figure S5. Pharmacological profiling of OCR of rabbit AF cells in the absence and presence of lactate. OCR of AF cells (A) were measured by Seahorse XFe96 Extracellular Flux Analyzer at basal conditions and with serial administration of 1 μM oligomycin, 0.3 μM FCCP, 100 mM 2DG and 1 μM rotenone. OCR was calculated and normalized to protein amount and the results were expressed as a mean of four different samples ± SEM. Individual parameters of OXPHOS (B) were derived from OCR profiles of AF cells ± lactate, as described in Materials and Methods. Addition of lactate did not significantly affect respiration reserved capacity (Res Cap), respiration total capacity (Tot Cap), non-glucose respiration (NG OCR) and non-mitochondrial oxygen consumption (NMR). Results are expressed as mean of four different samples (derived from four rabbits) ± SEM, * p < 0.05. Figure S6. Exogenously added lactate decreases matrix protein synthesis. Rabbit NP cell cultures exposed to physiological concentration of glucose (1mM) in the presence or absence of 4mM lactate. The presence of lactate decreases overall proteoglycan synthesis (35S-sulfate incorporation, p= 0.64, A), collagen matrix synthesis (3H-Lproline incorporation & collagenase sensitive fraction, p=0.21, B), and protein synthesis (3H-L-proline incorporation, p=0.1, C). D) Rabbit AF cells grown at 5% O2 in 5mM glucose, 5mM lactate, or 1mM glucose + 4mM lactate generated similar levels of total protein (black bars) and collagen (grey bars) synthesis. Results are expressed as mean of four different samples (derived from four rabbits) ± SEM. Figure S7. HK2 protein expression in AF and NP tissues. Western blot of hexokinase 2 (HK2) in rabbit AF and NP tissue protein extract (A) and their protein levels were quantified by normalizing to β-actin as loading control (B). Data are means ± SD of three independent experiments (three rabbits). * p<0.05.