Lactate preconditioning promotes a HIF-1α-mediated metabolic shift from OXPHOS to glycolysis in normal human diploid fibroblasts

Recent evidence has emerged that cancer cells can use various metabolites as fuel sources. Restricting cultured cancer cells to sole metabolite fuel sources can promote metabolic changes leading to enhanced glycolysis or mitochondrial OXPHOS. However, the effect of metabolite-restriction on non-transformed cells remains largely unexplored. Here we examined the effect of restricting media fuel sources, including glucose, pyruvate or lactate, on the metabolic state of cultured human dermal fibroblasts. Fibroblasts cultured in lactate-only medium exhibited reduced PDH phosphorylation, indicative of OXPHOS, and a concurrent elevation of ROS. Lactate exposure primed fibroblasts to switch to glycolysis by increasing transcript abundance of genes encoding glycolytic enzymes and, upon exposure to glucose, increasing glycolytic enzyme levels. Furthermore, lactate treatment stabilized HIF-1α, a master regulator of glycolysis, in a manner attenuated by antioxidant exposure. Our findings indicate that lactate preconditioning primes fibroblasts to switch from OXPHOS to glycolysis metabolism, in part, through ROS-mediated HIF-1α stabilization. Interestingly, we found that lactate preconditioning results in increased transcript abundance of MYC and SNAI1, key facilitators of early somatic cell reprogramming. Defined metabolite treatment may represent a novel approach to increasing somatic cell reprogramming efficiency by amplifying a critical metabolic switch that occurs during iPSC generation.


Defined metabolite treatment alters the metabolism of normal human fibroblast cells.
To determine if normal cells were capable of using alternative fuel sources in the absence of glucose, we investigated the impact of restricted fuel source availability on human foreskin dermal fibroblast (BJ) cell metabolism. To this end, BJ fibroblasts were initially cultured in medium containing 20 mM glucose, pyruvate, or lactate as the sole metabolite fuel source for 24 h. We first examined the impact of metabolite restriction on the protein and transcript abundance of metabolic enzymes. Pyruvate dehydrogenase (PDH) catalyses the conversion of pyruvate to acetyl-CoA, ultimately facilitating ATP production by OXPHOS 37 . Phosphorylation of PDH by PDK1 results in inhibition of PDH activity and renders cells more dependent on glycolysis to meet their energy needs 38 . Pyruvate kinase is the enzyme responsible for catalysing the conversion of phosphoenolpyruvate (PEP) to pyruvate, the last step in glycolysis 38,39 . Pyruvate kinase muscle isozyme 2 (PKM2) is an alternatively spliced isozyme of pyruvate kinase that, following phosphorylation, can translocate to the nucleus and facilitate increased transcription of enzymes that favour lactate production and glycolysis 40 . Immunoblot analysis revealed that BJ cells restricted to medium containing only glucose as a fuel source exhibited a significantly increased ratio of ser 232 -PDH to total PDH (p < 0.001) compared to cells cultured under control conditions. In contrast, BJ cells restricted to pyruvate or lactate as a fuel source exhibited a significantly decreased ratio of ser 232 -PDH to total PDH (p < 0.0001) compared to control (Fig. 1a). However, none of the metabolite-restricted media altered PKM2 or PDK1 protein levels (Fig. 1a). Interestingly, both pyruvate-and lactate-treated fibroblast cells exhibited significantly increased transcript abundance of hexokinase 2 (HK2) (p < 0.01), which enocodes the protein that catalyses the first step in glycolysis, compared to control (Fig. 1b) 39 . Furthermore, only lactate-treated BJ fibroblasts exhibited significantly increased transcript abundance of PDK1 (p < 0.05) and phosphoglycerate kinase 1 (PGK1) (p < 0.05), with a small but non-significant increase in lactate dehydrogenase A (LDHA) and PKM transcript abundance compared to control (Fig. 1b). In contrast, pyruvate-treated BJ fibroblasts exhibited significantly decreased glyceraldehyde 3-phosphate dehydrogenase (GADPH) (p < 0.05) transcript abundance compared to control (Fig. 1b). While GAPDH and PGK1 catalyse the sixth and seventh step of glycolysis, respectively, LDHA converts pyruvate to lactate at the end of glycolysis 39,41 . Defined metabolite treatment had no effect on the transcript abundance of tricarboxylic acid (TCA) cycle genes, ATP citrate lyase (ACLY), isocitrate dehydrogenase 1 (IDH1), oxoglutarate dehydrogenase (OGDH), malate dehydrogenase (MDH1), and succinate dehydrogenase complex iron sulphur subunit B (SDHB) (see Supplementary Fig. S1). These initial findings suggest defined metabolite treatment primarily impacts glycolytic enzymes rather than OXPHOS.
To validate the real time effect of defined metabolite treatment on BJ cell metabolism, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured by the glycolysis stress test and the mitochondrial stress test respectively (Fig. 2a). Cells treated with different metabolites exhibited similar basal glycolysis, glycolytic capacity and maximal respiration (Fig. 2b,c). However, lactate-treated BJ cells exhibited a significantly greater glycolytic reserve compared to pyruvate-treated cells (p < 0.05) (Fig. 2b). While lactate-treated BJ cells also exhibited significantly greater basal respiration (p < 0.01) than pyruvate-treated cells, pyruvate-treated BJ fibroblasts exhibited a significantly greater spare respiratory capacity than lactate-treated cells (p < 0.05) (Fig. 2c). These results suggest that lactate-treated BJ fibroblasts exhibit a bivalent metabolism based on their ability to switch to glycolysis when glucose becomes available.
In light of the observation that lactate-treated BJ fibroblasts became glycolytic upon injection with glucose and pharmacological inhibition of ATP synthase during the glycolysis stress test, we explored if this effect was sustained over a longer period. Due to the toxicity elicited by 24 h lactate treatment (Fig. 3a, left panel), we set out Figure 1. Defined metabolite treatment promotes post translational and transcriptional changes in human fibroblasts. BJ fibroblasts were cultured in defined metabolite media for 24 h prior to protein harvest and RNA isolation. (a) Immunoblots were probed with antibodies directed against the indicated metabolic markers for glycolysis and OXPHOS. Densitometric analysis of the ratio of ser 232 -PDH to total PDH band intensities normalized to β-Actin, revealed that BJ cells treated with glucose promoted significantly increased phosphorylation of PDH (indicative of glycolysis), whereas treatment with pyruvate or lactate resulted in significantly decreased phosphorylation of PDH (indicative of OXPHOS) compared to control-treated cells. Densitometric analysis of PKM2 and PDK1 band intensities normalized to β-Actin, revealed that 24 h defined metabolite treatment did not alter PKM2 or PDK1 protein abundance in BJ cells compared to control conditions. (b) qRT-PCR using ACTB and RPL37A as housekeeping genes, revealed that lactate-treatment significantly increased transcription of genes encoding the glycolytic enzymes, HK2, PGK1 and PDK1 compared to control. Pyruvate treatment resulted in a significant increase and decrease in the transcript abundance of genes enocding HK2 and GADPH, respectively, compared to control. The data presented represent N = 3 ± s.e.m. All qRT-PCR was performed in triplicate. The immunoblots are representative of three independent experiments. Full length blots can be found in Supplementary Fig. S4. Asterisks indicate significant difference (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****) and ns = no difference tested by One-way ANOVA and Dunnett's multiple comparisons test.
www.nature.com/scientificreports www.nature.com/scientificreports/ to determine the minimum about of time BJ fibroblast cells can be cultured in lactate-only medium prior to being switched into glucose-only medium and still exhibit a metabolic shift. Fibroblasts were cultured in glucose or lactate medium for 12, 16, 20 and 24 h prior to 48 h culture in glucose medium. Immunoblot analysis of the ratio of ser 232 -PDH to total PDH was used as an indicator for glycolytic metabolism. Densitometric analysis revealed that 20 h lactate pre-treatment significantly increased the ratio of ser 232 -PDH to total PDH (p < 0.01) compared to BJ cells cultured only in glucose medium (see Supplementary Fig. S2).
Defined metabolite treatment alters cell viability in a ROS-dependent manner. Glucose is the typical fuel source for most normal somatic cell types maintained in vitro, thus we sought to examine the impact of metabolite restriction on BJ cell growth and viability. Control-and glucose-treated cells exhibited similar growth over 72 h whereas pyruvate (p < 0.05)-and lactate-treated (p < 0.01) cells exhibited significantly decreased cell growth and elevated cell death within 24 h (Fig. 3a, left panel). Restricting cells to pyruvate as the sole fuel source strongly directs cellular metabolism to OXPHOS for ATP production 18 . A by-product of OXPHOS is mitochondrial ROS production 31 . While ROS are important signalling molecules, ROS build-up can cause cell death 32 . To examine if pyruvate-and lactate-induced cell death was a result of ROS build-up, the antioxidant precursor, N-acetyl-cysteine (NAC) was added to metabolite restricted media. Indeed, 24 h and 48 h of NAC Figure 2. Lactate treatment promotes bivalent metabolism in fibroblasts. BJ fibroblast cells were cultured in defined metabolite media for 24 h prior to analysis with the Seahorse XF e 24 Flux Analyzer. (a) Extracellular acidification rate (ECAR) normalized to total protein was used as proxy measure of glycolytic activity following subsequent injections of glucose, oligomycin and 2-deoxy-D-glucose (2-DG) during the glycolysis stress test. Oxygen consumption rate (OCR) normalized to total protein was used as a proxy measure of OXPHOS following subsequent injections of oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and antimycin A/rotenone (AA/RT) during the mitochondrial stress test. (b) No difference in basal glycolysis or glycolytic capacity was observed following glucose and oligomycin injection, respectively. However, lactate-treated BJ cells exhibited a significantly greater glycolytic reserve than pyruvate-treated cells. (c) Basal respiration was significantly elevated in lactate-treated BJ fibroblast cells compared to pyruvate-treated cells. However, lactate-treated BJ cells exhibited significantly lower spare respiratory capacity than pyruvate-treated cells. Maximal respiration did not differ between treatments. The data presented represent N = 4 ± s.e.m. with 5 technical replicates per treatment. Asterisks indicate significant difference (p < 0.05 = *, p < 0.01 = **) and ns = no difference tested by One-way ANOVA and Tukey's multiple comparisons test. exposure resulted in significantly increased viability of pyruvate-(p < 0.05) and lactate-treated (p < 0.05) fibroblast cells, respectively. (Fig. 3a, centre and right panels). These results suggest that pyruvate-and lactate-induced toxicity is caused, in part, by increased ROS.
To confirm that pyruvate and lactate treatment induce ROS build-up, BJ fibroblasts were cultured in defined metabolite medium for 20 h prior to live cell fluorescent quantification of ROS levels. Quantification of fluorescence intensity following staining with whole cell ROS indicator, CM-H2DCFDA, revealed that lactate-treated cells produced significantly more ROS than all other conditions (p < 0.0001) (Fig. 3b). Furthermore, NAC exposure significantly attenuated ROS production in BJ fibroblasts cultured in lactate medium (p < 0.0001) (Fig. 3b). Staining for mitochondrial ROS levels using MitoTracker CM-H2XRos revealed that mitochondrial ROS production was significantly reduced by NAC exposure in control-(p < 0.01), pyruvate-(p < 0.05) and lactate-treated (p < 0.01) BJ cells (Fig. 3c). A small but non-significant decrease in ROS levels was also observed in glucose-treated BJ cells supplemented with NAC (Fig. 3c). and ns = no difference tested by One-way ANOVA and Dunnett's multiple comparisons test as well as an Unpaired Two-tailed student's t-test. (b) BJ fibroblast cells were cultured in defined metabolite medium containing glucose, pyruvate or lactate as the sole fuel source with and without 1 mM NAC for 20 h. Live cell staining with the fluorescent cellular ROS indicator, CM-H2DCFDA (green), was performed. ImageJ analysis revealed that lactate-treated BJ cells produced significantly more ROS than all other treatments. NAC exposure significantly attenuated ROS production in lactate-treated cells. (c) Live cell staining with the fluorescent mitochondrial ROS indicator, MitoTracker CM-H2XRos (red), was performed. ImageJ analysis revealed that NAC significantly attenuated ROS production in control-, pyruvate-, and lactate-treated cells. A small but non-significant decrease in ROS levels was also observed in glucose-treated BJ cells supplemented with NAC. Nuclei within all cells were counterstained with Hoescht dye (blue). Scale bars = 100 μm. The fluorescence images presented are representative of at least three independent experiments. Data presented represent N = 3-4 ± s.e.m. with 3 technical replicates per treatment. Asterisks indicate significant difference (p < 0.05 = *, p < 0.01 = **, p < 0.0001 = ****) and ns = no difference tested by One-way ANOVA and Tukey's multiple comparisons test. (2020) 10:8388 | https://doi.org/10.1038/s41598-020-65193-9 www.nature.com/scientificreports www.nature.com/scientificreports/ Lactate promotes increased HIF-1α protein abundance in fibroblasts in a RoS-dependent manner. To gain mechanistic insight into the lactate-induced metabolic switch from OXPHOS to glycolysis, we examined the protein abundance of the transcription factor, HIF-1α. HIF-1α is a master regulator of glycolysis that promotes the transcription of several genes involved in glucose uptake and breakdown 38 . Under normoxic conditions, HIF-1α is translated and rapidly degraded in the cytosol 42 . Previous studies using cultured cancer cells have shown that lactate exposure results in stabilization of HIF-1α in normoxia 15 . To determine if lactate-treatment affects HIF-1α levels in non-transformed cells, fibroblasts were cultured in defined metabolite conditions for 20 h. BJ cells were cultured in DMEM under normoxic (20% O 2 ) and hypoxic (1% O 2 ) conditions as negative and positive controls respectively. Both pyruvate-(p < 0.01) and lactate-treated (p < 0.0001) BJ fibroblasts exhibited significantly increased HIF-1α stabilization compared to the negative control (Fig. 4a). Studies have shown that excess ROS can inhibit HIF-1α degradation under normoxic conditions 43 . To determine if lactate-or pyruvate-mediated HIF-1α stabilization was associated with ROS production, the antioxidant NAC was added to media containing only pyruvate or lactate as a fuel source. Indeed, NAC significantly attenuated HIF-1α stabilization in both pyruvate (p < 0.0001) and lactate-treated (p < 0.05) BJ cells. (Fig. 4b,c). Interestingly, NAC had a more pronounced inhibitory effect on HIF-1α stabilization in pyruvate-treated BJ cells compared to lactate-treated cells. Thus, lactate-treatment promotes stabilization of HIF-1α in BJ cells, in part, through a ROS related mechanism.
HIF-1α accumulation primes fibroblasts to switch from OXPHOS to glycolysis by increasing the abundance of PDK1 and PKM2 proteins. Previous studies have shown that culturing BJ cells in hypoxic conditions renders them more glycolytic 29 . To confirm the upregulation of glycolytic proteins downstream of HIF-1α, we cultured BJ fibroblasts in DMEM under normoxic (20% O 2 ) and hypoxic (1% O 2 ) conditions for 20 h prior to protein harvest (Fig. 5a). PDK1 (p < 0.001) and LDHA (p < 0.05) protein levels were significantly higher in BJ cells grown in hypoxia for 20 h compared to normoxic culture conditions (Fig. 5b). However, PKM2 protein levels were unaffected by hypoxia (Fig. 5b). In order to determine if the lactate-induced switch to glycolytic metabolism was sustained over longer periods, we compared the effect of pre-treating BJ cells in either glucose-or lactate-only medium for 20 h, followed by 48 h exposure to medium containing only glucose as a fuel source. We . Pyruvate and lactate-treated fibroblasts exhibit increased HIF-1α stabilization under normoxic conditions, which is attenuated by NAC exposure. BJ fibroblast cells were cultured in defined metabolite medium containing glucose, pyruvate or lactate as the sole fuel source. BJ cells cultured in DMEM in normoxia (20% O 2 ) or hypoxia (1% O 2 ) for 20 h were used as negative and positive controls, respectively. (a) Immunoblot analysis revealed that HIF-1α protein levels were significantly increased in pyruvate-and lactate-treated BJ cells under normoxic conditions compared to the negative control. BJ cells were cultured in defined metabolite medium containing pyruvate or lactate as the sole fuel source with or without 1 mM NAC for 20 h in normoxic conditions. (b,c) Immunoblot analysis revealed that HIF-1α levels were lower in BJ cells treated with NAC after 20 h in both pyruvate and lactate medium. The immunoblots are representative of at least three independent experiments. Full length blots can be found in Supplementary Fig. S4. The data presented represent N = 3-5 ± s.e.m. Asterisks indicate significant difference (p < 0.05 = *, p < 0.01 = **, p < 0.0001 = ****) and ns = no significant difference tested by One-way ANOVA and Dunnett's multiple comparison's test and an Unpaired Two-tailed student's t-test. www.nature.com/scientificreports www.nature.com/scientificreports/ Densitometric analysis revealed that PDK1 and PKM2 protein levels were significantly upregulated in response to lactate-pre-treatment, whereas LDHA was not. The immunoblots presented are representative of four independent experiments. Full length blots can be found in Supplementary Fig. S4. The data presented represent N = 4 ± s.e.m. Asterisks indicate significant difference (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***) and ns = no difference tested by an Unpaired Two-tailed student's t-test. www.nature.com/scientificreports www.nature.com/scientificreports/ found that PDK1 (p < 0.05) and PKM2 (p < 0.01) protein levels were significantly increased in lactate pre-treated cells compared to glucose-only treated cells (Fig. 5c,d). LDHA levels did not differ between treatments (Fig. 5d).
To confirm that elevated PDK1 and PKM2 protein levels observed in the lactate pre-treated cells were related to HIF-1α stabilization, BJ cells were cultured in glucose or lactate medium with and without KC7F2, a pharmacological inhibitor of HIF-1α 44 . Due to the combined toxicity of KC7F2 and lactate, BJ fibroblasts were only cultured in lactate medium supplemented with 20 μM KC7F2 for 12 h instead of 20 h. We confirmed that 12 h treatment with 20 μM KC7F2 was sufficient to significantly reduce HIF-1α protein levels in BJ cells (p < 0.01) (see Supplementary Fig. S3). Upregulation of PDK1 (p < 0.001), PKM2 (p < 0.05) and LDHA (p < 0.01) induced by 12 h culture in hypoxic conditions was significantly attenuated by KC7F2 treatment (Fig. 6a). In contrast, PDK1, PKM2 and LDHA protein levels were unchanged in fibroblasts pre-treated with glucose medium in the presence of KC7F2 for 12 h (Fig. 6b). However, PDK1 and PKM2 levels were significantly lower (p < 0.05) in fibroblasts pre-treated with lactate in the presence of KC7F2 for 12 h (Fig. 6c). These findings support our claim that lactate pre-treatment primes BJ fibroblast cells to upregulate glycolytic enzymes in a HIF-1α-dependent manner.
Lactate preconditioning upregulates the transcript abundance of MYC and SNAI1. Recent studies have demonstrated that c-MYC promotes a hyperenergetic state during early reprogramming to facilitate optimal iPSC generation 27 . Estrogen related receptor alpha (ERRα) and it's cofactor, peroxisome proliferator-activator receptor gamma coactivator 1-beta (PGC1-β), are also implicated in the acquisition of this hyperenergetic state 36 . In addition to a metabolic switch, somatic cells must undergo a mesenchymal-to-epithelial transition during reprogramming 45,46 . Although snail family transcriptional repressor 1 (SNAIL) is a mediator of epithelial-to-mesenchymal transition (EMT), it is paradoxically essential to early somatic cell reprogramming 45,46 . To gauge the impact of lactate pre-treatment on these markers of early reprogramming, BJ cells were cultured in glucose or lactate medium for 20 h prior to 48 h cultivation in glucose-only medium. Significantly increased transcript abundance of both MYC (p < 0.05) and SNAI1 (p < 0.01), but not ESRRA or PPARGC1B, was observed in lactate pre-treated fibroblast cells compared to cells cultured only in glucose medium (Fig. 7). These findings suggest that lactate production may regulate expression of specific genes involved in early somatic cell reprogramming.

Discussion
In this study, we demonstrated that pre-treating human fibroblast cells with culture medium containing lactate as the sole fuel source, facilitates a metabolic switch from OXPHOS to glycolysis, in part, through ROS-mediated stabilization of HIF-1α. Specifically, we observed that BJ fibroblasts cultured in medium containing lactate or pyruvate as a fuel source for 24 h exhibited significantly reduced phosphorylation of PDH. Conversely, fibroblasts cultured in glucose-containing medium for 24 h displayed elevated PDH phosphorylation, a marker of glycolytic metabolism 38,47 . These findings are consistent with a previous study in which HeLa cells grown under glucose-only culture conditions exhibited reliance on aerobic glycolysis 18 . However, when cultured in pyruvate-only medium, HeLa cells switched to OXPHOS to facilitate ATP production 18 . Interestingly, despite glucose deprivation, lactate-treated BJ cells unexpectedly exhibited increased transcript abundance of several genes encoding proteins responsible for catalysing steps of the glycolytic pathway. A recent study by Zhang et al. discovered that lactate acts as an epigenetic regulator by inducing histone lactylation in a dose-dependent manner 48 . Indeed, the onset of aerobic glycolysis or hypoxia-induced glycolysis directly correlated with both increased lactate production and histone lactylation, and direct induction of glycolytic gene expression 48 . Therefore, it is possible that in our model, lactate medium transcriptionally primes human fibroblasts for glycolytic metabolism pending substrate availability. Earlier studies have demonstrated that cancer cells are capable of using lactate as their preferred fuel source 14,17 . Furthermore, a study by Hui et al. revealed that lactate is the primary fuel source for the TCA cycle in most tissues and tumours 13 .
Metabolic flux, as assessed by Seahorse analysis, further supported our theory that lactate treatment primes BJ cells for glycolytic metabolism. BJ fibroblasts pre-treated with lactate medium exhibited a significantly greater glycolytic reserve and significantly lower spare respiratory capacity compared to fibroblasts pre-treated with pyruvate medium. Recent studies using primary human dermal fibroblasts showed that mitochondrial spare respiratory capacity negatively correlates with somatic cell reprogramming efficiency as well as pluripotency 37,49 . Interestingly, we found that lactate pre-treatment resulted in greater BJ cell basal respiration compared to pyruvate pre-treatment. Elevated basal respiration and reduced spare respiratory capacity in lactate pre-treated fibroblasts implies that these cells were respiring at their maximum capacity even prior to induced ETC uncoupling. c-MYC induces a hyperenergetic metabolic state during reprogramming that is necessary for the transition to pluripotency 27 . Our findings demonstrate that while both pyruvate and lactate treatment result in reduced PDH phosphorylation, only lactate pre-treatment promotes a hyperenergetic bivalent metabolic state.
Although BJ fibroblasts were capable of using lactate or pyruvate as fuel sources, both metabolites promoted an inhibition of cell growth and elevated cell death. Cell growth is maintained by the production of anabolic precursors such as ribose-5-phosphate (R5P), in a manner largely dependent on the pentose phosphate pathway (PPP) 50,51 . In addition to providing R5P, the PPP also produces nicotinamide adenine dinucleotide phosphate (NADPH), a key metabolic product that provides the reducing power to fuel protein-based antioxidant systems and recycle oxidized glutathione 51 . Since PPP activity relies on glycolytic flux, it is possible that restricting fibroblasts to lactate or pyruvate as a fuel source results in a deprivation of vital upstream glycolytic intermediates within the PPP that would otherwise support proliferation and antioxidant systems. Supplementation with NAC, a precursor to the antioxidant, glutathione, improved the viability of pyruvate-and lactate-treated BJ fibroblast cells. These findings suggest that pyruvate and lactate treatments elicit cytotoxicity via oxidative stress caused by excess ROS production and/or insufficient antioxidant synthesis 52  www.nature.com/scientificreports www.nature.com/scientificreports/ To determine if lactate pre-treatment upregulates PDK1 and PKM2 in a HIF-1α dependent manner, BJ cells were cultured in either glucose or lactate medium for 12 h with and without the HIF-1α inhibitor, KC7F2, prior to 48 h culture in glucose medium under normoxic (20% O 2 ) conditions. Immunoblot analysis of downstream metabolic target of HIF-1α was performed. (b) Densitometric analysis revealed that PDK1, PKM2 and LDHA protein levels were unaffected by KC7F2 in BJ cells pre-treated with glucose. (c) However, BJ cells pre-treated with lactate exhibited significantly lower protein levels of PDK1 and PKM2 in the presence of KC7F2 compared to lactate pre-treatment alone. The immunoblots presented are representative images of three independent experiments. Full length blots can be found in Supplementary Fig. S4. The data presented represent N = 3 ± s.e.m. Asterisks indicate significant difference (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***) and ns = no significant difference tested by and Unpaired Two-tailed student's t-test.
While peroxisomes and the endoplasmic reticulum are organelles capable of generating cellular ROS, mitochondria are the major site of ROS production in mammalian cells 53 . ROS exit the mitochondria and enter the cytosol by diffusion or passage through voltage dependent anion channels (VDAC) 54 . Under oxidative stress conditions, a growing body of work has revealed that mitochondrial ROS activates transient openings of voltage-gated mitochondrial permeability transition pore (mPTP) channels 55,56 . When open, matrix metabolites, such as ROS, exit the mitochondria through mPTPs and enter the cytosol [54][55][56] . This effect has been coined ROS-induced ROS release (RIRR) 55,57 . Cytosolic ROS has the potential to react with redox-sensitive molecules, activate redox-sensitive signalling pathways and induce RIRR in proximal mitochondira 55 . For example, during the Fenton reaction, hydrogen peroxide (H 2 O 2 ) reacts with ferrous iron (Fe 2+ ) to generate ferric iron (Fe 3+ ), a hydroxyl radical (•OH), and a hydroxyl ion (OH − ) 58 . Transient ROS-induced mPTP openings are associated with early phase somatic cell reprogramming and their metabolic switch 35,59 . Studies in human and mouse fibroblast cells have shown that mPTP openings promote demethylation of pluripotency promoters, an integral event in the acquisition of pluripotency during the later stages of reprogramming 35,59 . Indeed, increasing mPTP opening frequency with ROS-inducing agents prior to the metabolic switch was shown to increase reprogramming efficiency 35 . Our findings demonstrate that while pyruvate-and lactate-treated fibroblasts do not differ in their production of mitochondrial ROS after 20 h, lactate-treated cells exhibit significantly greater total cellular ROS levels. It is possible that lactate promotes mPTP openings, accelerating the release of ROS into the cytosol. Furthermore, lactate has been shown to propagate ROS production via the Fenton reaction by forming a complex with Fe 3+ that reacts with H 2 O 2 to produce additional •OH 60 . As such, we are currently investigating the impact of lactate medium on ROS-induced mPTP openings.
As many tumours exist in hypoxic environments, it is not surprising that they exhibit high levels of HIF-1α 62 . However, various cellular conditions exist which allow for HIF-1α accumulation in normoxia 15,43,63 . For example, ROS stabilizes HIF-1α under normoxic conditions by oxidizing Fe 2+ to Fe 3+ , thereby rendering PHD inactive 64,65 . Further evidence has emerged that lactate can inhibit PHD activity through its conversion to pyruvate which competitively inhibits α-KG from associating with PHD 15 . In this study we showed that both pyruvate and lactate are capable of stabilizing HIF-1α protein levels under atmospheric oxygen. However, NAC treatment only partially attenuated lactate-induced HIF-1α stabilization whereas pyruvate-induced HIF1α stabilization was almost entirely ablated by NAC exposure. These findings suggest that while both pyruvate and lactate can facilitate HIF-1α stabilization through ROS production, lactate may further directly stabilize HIF-1α in a ROS-independent manner. It is also possible that lactate-induced histone lactylation indirectly contributes to lactate-mediated HIF-1α stabilization in BJ fibroblast cells. Although these are plausible mechanisms to explain our finding that exogenous lactate increases the transcript abundance of genes encoding several glycolytic enzymes, further studies are warranted to explore this relationship.
In this study we showed that lactate pre-treatment significantly increased PDK1 and PKM2 protein levels in BJ cells through a HIF-1α-dependent mechanism. Small molecule activation of HIF-1α during somatic cell Figure 7. Pre-treatment of fibroblasts with lactate promotes increased transcript abundance of MYC and SNAI1. BJ fibroblast cells were cultured in either glucose or lactate medium for 20 h followed by 48 h culture in glucose medium under normoxic conditions prior to RNA extraction. qRT-PCR analysis using HPRT1 and RPL37A as housekeeping genes revealed that MYC and SNAI1 transcript abundance was significantly increased in response to lactate pre-treatment. In contrast, ESRRA and PPARGC1B transcript abundance were no different between treatments. The data presented represent N = 3 ± s.e.m. with 3 technical replicates per treatment. Asterisks indicate significant difference (p < 0.05 = *, p < 0.01 = **) and ns = no significant difference tested by an Unpaired Two-tailed student's t-test. www.nature.com/scientificreports www.nature.com/scientificreports/ reprogramming has been shown to dramatically increase fibroblast reprogramming efficiency by upregulating PDK1 and PKM2 38 . It is possible that lactate promotes HIF-1α stabilization by inhibiting PHD activity through competitive inhibition of α-KG and by propagating the Fenton reaction (Fig. 8). Furthermore, as NRF2 has been shown to act upstream of HIF-1α, it is possible that lactate-mediated ROS production initiates the NRF2 cell stress response pathway to further upregulate HIF-1α 34 (Fig. 8). However, further studies are warranted to discern the impact of lactate treatment on NRF2 activity in BJ fibroblasts.
In addition to lactate pre-treatment promoting increased PKM2 and PDK1 protein abundance, we demonstrated that this pre-treatment strategy results in increased transcript abundance of SNAI1 and MYC. Elevated SNAI1 transcript abundance following lactate exposure is in line with a study conducted in lung cancer cells which demonstrated that that lactate promotes SNAI1 expression in a dose-dependent fashion 66 . With respect to MYC, recent focus has shifted towards its endogenous role. Exogenous expression of MYC is considered a dispensable Yamamaka factor that can be replaced by overexpression of PDK1 and PKM2, various chemicals, or other enhancer factors such as NANOG and LIN28 25,26,30,38 . Prieto et al. demonstrated that endogenous c-MYC is fundamental to early stage reprogramming events such as mitochondrial remodelling and activation of glycolysis 27 . It is therefore possible that the lactate-induced metabolic shift from OXPHOS to glycolysis observed in this study is in part mediated by endogenous c-MYC. Interestingly, other markers for early stage reprogramming, including ESRRA and PPARGC1B, where not affected by lactate exposure. It is possible that lactate exposure promotes lactylation of specific histone lysine residues and selectively induces expression of certain pluripotency Figure 8. Proposed mechanism of action for lactate-induced upregulation of glycolytic metabolism in normal human diploid fibroblasts. Extracellular lactate is taken up by monocarboxylic acid transporters and can then be converted to pyruvate to fuel the tricarboxylic acid (TCA) cycle, which produces reducing agents that support electron transport chain (ETC)-mediated adenosine triphosphate (ATP) production by oxidative phosphorylation (OXPHOS). The mitochondrial reactive oxygen species (ROS) produced by ETC activity translocate to the cytosol freely, through voltage dependent anion channels (VDAC), or through open mitochondrial permeability transition pores. In the cytosol ROS inhibit prolyl hydroxylase (PHD) activity through the Fenton reaction which oxidizes ferrous iron (Fe 2+ ), a critical PHD cofactor, to ferric iron (Fe 3+ ). Following the inactivation of PHD, hypoxia inducible factor 1 alpha (HIF-1α) is no longer tagged for proteasomal degradation. Instead, HIF-1α translocates to the nucleus where it dimerizes with HIF-1β and binds to the hypoxia response element (HRE) initiating the transcription of glycolytic enzymes such as pyruvate dehydrogenase kinase 1 (PDK1) and pyruvate kinase muscle isozyme 2 (PKM2). Lactate is also capable of inhibiting PHD activity through its conversion to pyruvate which competitively inhibits PHD co-factor, α-ketoglutarate (α-KG), from associating with PHD. Lactate may also enhance ROS-mediated inhibition of PHD by the ROS induced ROS release (RIRR) effect. Lactate can form a complex with Fe 3+ which then reacts with the ROS generated from the Fenton reaction to propagate the production of more ROS. It is also possible that lactate-mediated ROS production promotes increased HIF-1α levels by activating nuclear factor erythroid 2-related factor 2 (NRF2). However, future studies are required to elucidate the role of NRF2 in lactate-mediated induction of glycolytic metabolism. (2020) 10:8388 | https://doi.org/10.1038/s41598-020-65193-9 www.nature.com/scientificreports www.nature.com/scientificreports/ genes. Future studies using chromatin immunoprecipitation with anti-lactyllysine antibodies 48 will help identify genes regulated by histone lactylation following exposure of human fibroblasts to exogenous lactate.
Mesenchymal to epithelial transition (MET) is another hallmark of reprogramming fibroblasts to iPSCs 45,46 . Counterintuitively, Unternaehrer et al. demonstrated that human and mouse fibroblasts cells expressing higher levels of endogenous SNAI1, an EMT regulator, actually exhibited more efficient reprogramming 45 . These researchers further postulated that SNAI1 expression increases reprogramming efficiency by inhibiting let-7 family members 45 . Let-7 is a family of tumour suppressors whose inhibition has been shown to promote reprogramming efficiency 67 . LIN28, a regulator of stem cell metabolism, is a known repressor of let-7 miRNA processing 68,69 . It is therefore possible that lactate pre-treatment has the potential to increase reprogramming efficiency not only by promoting the metabolic switch event, but also by mirroring LIN28-mediated repression of let-7 miRNA processing through increased SNAI1 expression.
In conclusion, our work demonstrates that short-term culturing of normal human dermal fibroblast cells in medium containing lactate as the sole metabolite fuel source primes BJ fibroblast cells to transition from OXPHOS to glycolysis metabolism. Indeed, fibroblasts cultured in lactate medium exhibit increased ROS production which, in part, contributes to the stabilization of HIF-1α and subsequent upregulation of glycolytic enzymes, PDK1 and PKM2. By promoting the transition from OXPHOS to glycolysis, lactate pre-treatment could serve as a novel approach to amplify the metabolic switch during the generation of iPSCs. Furthermore, by triggering HIF-1α-mediated upregulation of PDK1 and PKM2 as well as increased transcript abundance of MYC and SNAI1, lactate treatment may be able to eliminate the need for exogenous c-MYC during somatic cell reprogramming. We are currently in the process of exploring these hypotheses. Ultimately, the findings from this study may lead to the development of a safer and more efficient method of creating human iPSCs that can be utilized for pluripotent stem cell-based therapies. Defined metabolite media were prepared as follows. Base medium was prepared by dissolving DMEM powder lacking glucose, L-glutamine, sodium pyruvate and sodium bicarbonate (#5030; Sigma-Aldrich) in 1 L deionized/ double distilled water supplemented with 3.7 g/L sodium bicarbonate (#SX0320-1; EMD Millipore) and sterilized through a 0.1 μm filter. Immediately prior to experimentation, base media was supplemented with 4 mM L-glutamine (#17605-E; Lonza) and 10% FBS. FBS was dialyzed using regenerated cellulose dialysis tubing with a 3,500 Dalton cut-off (#21-152-9; Fisher Scientific) for 2 d in buffered base medium with one media change and sterilized through a 0.2 μm filter. Control, glucose, pyruvate and lactate defined metabolite media were prepared by adding 20 mM D-(+)-glucose (#G7021; Sigma-Aldrich) and 1 mM sodium pyruvate (#P2256; Sigma-Aldrich), 20 mM glucose, 20 mM sodium pyruvate and 20 mM sodium L-lactate (#71718; Sigma-Aldrich), respectively, to the supplemented base medium. A metabolite concentration of 20 mM was selected based on reports that the concentration of lactate in tumour microenvironments most commonly lies within the range of 10-30 mM 10,70 . Prior to treatment with defined metabolite medium, fibroblasts were washed twice with phosphate buffered saline (PBS) (#17-513F; Lonza) to remove traces of DMEM.