Rationale. This study is motivated by our new hypothesis postulating that lactate produce by NP glycolysis is not a waste end-product in the intervertebral disc but rather is an important and precious carbon source for OXPHOS in AF cells residing in a nutrient-poor disc environment. We also hypothesize that utilization of lactate by AF cells also serves to minimize NP lactate accumulation and its negative impact on NP cells. To investigate our postulated lactate-dependent metabolic symbiosis between NP and AF, we examined the capacity of AF cells to uptake and utilize lactate using in vitro AF cell and ex vivo disc organ culture model systems. Both radioactive and stable isotope tracing by HRMS were employed for this purpose. Rabbit models were used as they provide enough disc cells for cell cultures and their disc size is sufficiently large for the 13C-lactate injection and tracing study. Additionally, we decided to perform initial characterization of lactate metabolic symbiosis in young normal discs, which are readily available in rabbits but not in humans.
AF cells are less glycolytic than NP cells. Because we used rabbit nucleus pulposus (rNP) and annulus fibrosus (rAF) cell cultures as the model system in our study, we first needed to confirm that these cell types in our in vitro model exhibit metabolic features consistent with those previously reported (23, 26). Indeed, rNP cells grown in culture at 5% O2 readily consumed glucose (Fig. 1A), then produced and secreted lactate at a high steady-state rate (Fig. 1B). In contrast, AF cells grown under the same condition produced a much lower amount of lactate (Fig. 1D), but also consumed glucose at a much slower rate than NP cells (Fig. 1C) even though AF cells proliferate at similar or faster rates than NP cells under these conditions (23, 27, 28). These results suggest that AF cells are less glycolytic than NP cells, and that AF cells use less glucose than NP cells, likely because AF cells utilize OXHPOS which generates more ATP per glucose than glycolysis.
AF cells tolerate high lactate levels. Physiological lactate concentrations in human disc tissue from periphery to the center have been reported to range from 2 to 16 mM while glucose concentrations range from 1–5 mM (4). To determine if rAF cells in culture could tolerate lactate in this physiologic range, rAF cell cultures were exposed to media containing exogenously added lactate ranging from 0 to 20 mM lactate in the presence of physiologic glucose concentration (5 mM). Lactate up to 10 mM had no effects on AF cell viability in culture as assessed by CCK8 assay (Fig. S1. B, Supplementary Material). Only at high lactate (20 mM) concentration did we observed a modest decrease in AF cell viability by about 20%, consistent with the reported toxic effects of excessive acidity due to high lactate levels on disc cells (6). Lactate up to 10 mM also had no discernable effects on AF cell morphology or density in vitro (Fig. S1. A, Supplementary Material). Given that normal blood lactate concentration is 0.5-1 mM, these results suggest that AF cells have evolved to tolerate high lactate concentrations in disc tissue that are 5–10 times the level found in serum (5). Because AF physiologic lactate concentrations range mostly between 2–6 mM (4), we chose 4 mM lactate to test its effects on AF cell metabolism in all our subsequent experiments.
Lactate uptake by AF cells. Lactate tolerance by AF cells suggests utilization, but to do so AF cells must be able to import lactate from their extracellular environment. To determine whether AF cells can import lactate, we performed a radioactive tracing assay using 14C-lactate to measure cellular 14C uptake. Rabbit AF cells exposed to increasing 14C-lactate concentrations resulted in a proportional increase in 14C levels in the cells after the cells were extensively washed with PBS to remove nonspecific binding of 14C lactate (Fig. 2A). To be sure that uptake was not due to nonspecific attachment of 14C-lactate to cells or plastic surface of the culture plate, we also included no-cell and dead cell controls, e.g. AF cells killed with 40% ethanol. These control samples showed minimal radioactive counts (Fig. 2A), suggesting negligible nonspecific binding of 14C lactate to AF cells. HepG2 cells, a human hepatocyte carcinoma cell line known to import lactate, were also included as a positive control for our uptake assay which resulted in a 14C-lactate-concentration dependent increase in radioactive counts in the cell lysate, as expected (Fig. S2, Supplementary Material). The results from our 14C-lactate uptake assay demonstrated lactate import into AF cells.
Lactate import into cells is mediated preferentially via the monocarboxylate transporter 1 (MCT1) (29, 30). To determine if AF cells express MCT1, we performed qRT-PCR and found higher MCT1 mRNA expression in AF cells cultured in the presence of lactate (Fig. 2B). Additionally, AF but not NP tissue expresses an abundant amount of MCT1 protein (Fig. 2C). Our findings suggest that AF has the molecular machinery and capability to import lactate from the extracellular environment. In contrast, MCT4 protein, a known lactate exporter in hypoxic tissue (12, 31, 32), is expressed mostly in NP but not AF tissue (Fig. 2C). These data support the notion of lactate metabolic synergy between NP and AF tissues whereby NP produces and exports lactate via MCT4 into the extracellular space, which is then imported into AF cells via MCT1.
Lactate conversion to pyruvate by AF cells. Because AF cells reside within a more oxygenated region of the disc, we postulate that AF cells convert lactate back into pyruvate for its subsequent conversion to acetyl-coA to be shuttled in the TCA cycle for OXPHOS. To test this idea, we performed stable isotope tracing using 3-13C-lactate by HRMS. AF cell cultures labeled with 4 mM 3-13C-lactate for 24 hours resulted in 35 ± 4% atomic percent enrichment (APE) of M + 1 lactate and 19 ± 6% APE of M + 1 pyruvate (Fig. 3A). M + 1 indicates that one 13C carbon is present in these molecules. These findings confirmed that lactate is taken up and converted into pyruvate by AF cells in an in vitro cell culture model system.
To further demonstrate that AF cells in their native tissue are also capable of importing lactate and converting it to pyruvate, we performed stable isotope 13C-lactate labeling using an ex vivo disc organ culture model. Rabbit functional spine units (FSUs) containing vertebrae-disc-vertebrae were injected with 3-13C-lactate into the NP region to give an estimated final 3-13C-lactate concentration of ~ 5–10 mM. The FSUs were then incubated in the culture media for three days before being analyzed by HRMS. Under these conditions, there was a 28 ± 15% enrichment in M + 1 13C-lactate and 19 ± 9% APE in M + 1 pyruvate in AF tissue (Fig. 3B). As a control, we also incubated rabbit FSUs in culture media containing 4 mM 3-13C-lactate for three days which resulted in 68 ± 9% enrichment in M + 1 13C-lactate and 48 ± 7% APE in M + 1 pyruvate in AF tissue extract (Fig. S3, Supplementary Material). These findings demonstrated that AF cells in their native tissue environment can uptake and convert lactate into pyruvate.
Lactate dehydrogenase isozyme 1 (LDH1), a homo-tetramer of four H protein subunits, preferentially converts lactate to pyruvate (Fig. 3C) (33, 34). H is expressed significantly more in AF than NP tissue as shown by Western blot analysis (Fig. 3E), which is consistent with 13C being traced to pyruvate in our 13C-lactate tracing experiment. LDH5, an isozyme consisting of a homo-tetramer of four M protein subunits, preferentially converts pyruvate to lactate (33, 34). We expected the M protein to be expressed mostly in the hypoxic NP and less so in AF tissue. Surprisingly, this was not the case as M is expressed similarly in both NP and AF tissue (Fig. 3D), suggesting that AF cells possess as as much enzymatic capability as NP cells to convert pyruvate to lactate.
Although pyruvate can enter the triccyclic acidic (TCA) cycle through its conversion to oxoaloacetate by pyruvate carboxylase, pyruvate primarily enters the TCA cycle through its conversion to acetyl-coA (Fig. 6). The enzyme responsible for catalyzing the converion of pyruvate to acetyl-coA is pyruvate dehydrogenase (PDH). As expected, PDH is expressed three fold more in AF than NP tissue (Fig. 4A). Conversely, pyruvate dehydrogenase kinase 1 (PDK1), an enzyme that phosphorylates and inhibits PDH activity, is expressed about twofold more in NP than AF tissue (Figs. 4A). Together, these results are consistent in indicating that AF cells, much more so than NP cells in disc tissue, possess the molecular machinery necessary for importing lactate through MCT1, converting lactate to pyruvate by LDH1, and converting pyruvate to acetyl-coA by PDH.
Lactate conversion to TCA intermediates and amino acids by AF cells. Our 13C-lactate tracing experiment using a rabbit AF cell culture model also revealed that 13C was present in several tricarboxylic acid (TCA) intermediates, including succinate (14.5 ± 4.5% APE), fumarate (18 ± 5% APE), and malate (26 ± 4% APE) (Fig. 4B). Detection of 13C label in these three TCA metabolites, with succinate and fumarate being the precursors of malate, might be due to the unfavorable thermodynamic reaction of converting malate to oxaloacetate by malate dehydrogenase (ΔG = + 6.7 kcal mol−) (35), resulting in the buildup of these TCA intermediates compared to others (Fig. 6). Production of TCA cycle intermediates originated from lactate as well as the presence of lactate-handling enzymes in AF cells provide strong evidence to support the capability of AF cells to uptake and utilize lactate as a carbon source it for aerobic metabolism.
In addition to detecting heavy isotope labeling in the TCA intermediates, 13C was also traced to several amino acids in AF cell culture labeled with 13C-lactate. These include M + 1 glutamate (31 ± 5% APE), M + 1 glutamine (0.5 ± 0.2% APE), and M + 1 alanine (4.2 ± 0.2 APE) (Fig. 4B). These results suggest that AF cells can utilize lactate to make amino acids since alanine biosynthesis can be derived from pyruvate and both glutamine and glutamate can be enzymatically derived from α-ketoglutarate (36).
Using the same 13C-lactate tracing experiment, we also traced 13C to the malate and glutamate in human AF cell culture (Fig. S4A, Supplementary Material), and more importantly in rat AF tissues in vivo (Fig. S4B, Supplementary Material) to approximately 10% APE. Together, these findings demonstrated that AF cells can uptake and convert lactate to TCA intermediates and amino acids, and that this metabolic phenotype appears to be universal, i.e. not species specific, and occurs both in vitro cell culture and in vivo models.
Lactate oxidative phosphorylation by AF cells. Conversion of lactate into TCA intermediates by AF cells implies oxidative phosphorylation (OXPHOS) of lactate. However, 13C from 13C-lactate was also traced to amino acids, suggesting that lactate is used by AF cells for biosynthesis in addition to being used in OXPHOS to generate ATPs. To determine directly if lactate is used for OXPHOS, rabbit AF cells cultured in 1 mM glucose ± 4 mM lactate were analyzed using the Seahorse XFe96 Extracellular Flux Analyzer. Oxygen consumption rate (OCR), which reflects the extent of OXPHOS, was measured at basal conditions and following addition of specific inhibitors of the electron transport chain (Fig. S5A, Supplementary Material) (37). Under these conditions, lactate increased the basal OCR rate and the mitochondrial ATP-linked respiration in AF cells (Fig. 5A), but it did not have any significant effects on several individual parameters of OXPHOS, including reserve capacity, maximum total respiratory capacity, proton leak, non-glucose respiration, and nonmitochondrial oxygen consumption (Fig. S5B, Supplementary Material). These findings provide further evidence of lactate metabolism via OXPHOS to generate ATP in AF cells.
Lactate increases matrix synthesis in AF cells. Lactate increases OXPHOS and mitochondrial ATP-linked respiration, as well as production of amino acids in AF cells when they were grown under the physiological nutrient condition of low glucose (1 mM). A vital function of AF cells is to synthesize extracellular matrix, particularly the collagens, a process requires energy and amino acid building blocks. These observations raised a question of whether lactate can serve as a biofuel for matrix synthesis in AF cells. Indeed, our matrix synthesis assays using radioactive tracers revealed that AF cells synthesized almost twice as much as the total collagen (Fig. 5B, panel B.2) in the presence of 4 mM lactate than without when cells were grown in low glucose (1 mM) that mimic disc nutrient niche. Likewise, lactate stimulated total protein synthesis in AF cells to about 20% under the same condition (Fig. 5B, panel B.1). Interestingly, proteoglycan synthesis was also slightly increased in the presence of lactate but was not statistically significant (Fig. 5B, panel B.3). Together, these findings demonstrate that AF cells utilize lactate as a biofuel to produce matrix protein under physiologic glucose concentration. In contrast, lactate treatment of NP cell cultures decreased overall matrix synthesis in these cells, suggesting that NP cells do not metabolize lactate to make matrix (Fig. S6, Supplementary Material).