Cell stemness was maintained and metabolic reprogramming induced by hypoxia
Figure 1A shows the colony forming ability of human BM-derived EPCs under normoxia (~20% O2) and hypoxia (1% O2). Colony formation increased to 228±41% of the normoxic rate when hypoxic conditions were employed (p < 0.05). Expression of the stemness markers, Nanog, Oct4, Klf4 and Sox2, was higher under hypoxia than under normoxia (Fig. 1B), indicating more favorable maintenance of stem-like characteristics.
A Seahorse Real-time Extracellular XFe96 Flux Analyzer (14) was used to assess glycolytic function and mitochondrial respiration. Rates of glycolysis were increased to 366±74% (p < 0.001) and maximal glycolytic capacity to 145±19% (p < 0.05) under hypoxic compared with normoxic conditions (Fig. 1C). Rates of basal respiration decreased to about 30 ±6% and of maximal respiration to 8±2% (both p < 0.001) compared with normoxic conditions (Fig. 1D).
Relative enrichment of specific metabolites was analyzed by Liquid Chromatography-Mass Spectrometry (LC–MS) to define metabolic profiles and a heatmap for intermediates of glucose metabolism is presented in Figure 2A. Hypoxic conditions resulted in reduced D-glucose and increased glucose-6-P and pyruvate (all p<0.05), indicating a shift to greater glucose consumption and enhanced glycolysis (Fig. 2B). Levels of D-ribulose 5-P were also higher under hypoxia than under normoxia (p<0.05; Fig. 2C), indicating enhanced pentose phosphate pathway (PPP) activity. By contrast, levels of citrate, isocitrate, succinate, fumarate, malate and oxaloacetate (OAA) were all lower under hypoxia than under normoxia (all p<0.05, Fig. 2D), suggesting reduced TCA cycle activity under conditions of 1% O2.
13C glucose tracing
To ensure that the metabolites listed above originated from free glucose (rather than from gluconeogenesis, for example), stable isotope labeling experiments using 13C-labeled glucose were performed and isotopic enrichment quantified by High Pressure/Performance Liquid Chromatography-Q-Exactive-Mass Spectrometer (HPLC-QE-MS). No differences were found in 13C-labeled glycolytic intermediates, such as m+3 iso-topologues of glucose 6-P, phosphoenolpyruvate or pyruvate, between the two conditions (Fig. 2E). However, there was a significant increase in 13C-labeled intermediates of the PPP, such as m+4 iso-topologues of 6-phosphogluconate, ribose 5-P and sedoheptulose 7-P (all p<0.05), in hypoxic cells (Fig. 2F). Moreover, decreased 13C-labeling of TCA cycle intermediates, such as m+2 iso-topologues of citrate, succinate and malate (all p<0.05), was seen under hypoxia, suggesting a reduced contribution of glucose carbon to TCA cycle activity (Fig. 2G). Non-glucose-derived metabolites (m+0 iso-topologues) of glycolysis, PPP and TCA cycle showed no significant differences between the two conditions. The above findings indicate greater flow of glucose carbon to the PPP and reduced flow to the TCA cycle under hypoxic conditions.
Effects of glycolytic inhibition on EPC proliferation
Key enzymes of pathways of glucose metabolism were inhibited and effects on cell stemness assessed.
Bromopyruvate acid (BP) was used to inhibit hexokinase II (HK II), the 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB3) inhibitor, compound 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), to reduce fructose-2,6-bisphosphate (Fru-2,6-BP) levels and inhibit phosphofructokinase (PFK) and PKM2-IN-1 to inhibit pyruvate kinase (PKM2) (15-17). BP reduced EPC proliferation under both hypoxic and normoxic conditions and colony forming capacity was almost totally abolished by 100uM BP (Fig. 3B). Similar results were found with 3PO (Fig. 3C) and PKM2-IN-1 (Fig. 3D). These data demonstrate that glycolysis is essential for EPC proliferation.
Effects of PPP inhibition on EPC proliferation
PPP activity is vital for anabolic biosynthesis and anti-oxidant defense (18) and depends on the diversion of glucose carbon away from the glycolytic pathway. Glucose-6-phosphate dehydrogenase (G6PD) is considered to be a key rate-controlling enzyme of the PPP and can be competitively inhibited by 6-AN (19). 6-AN reduced EPC proliferation in a dose-dependent manner both under normoxia and under hypoxia (Fig. 4A) and colony formation in the presence of 20uM 6-AN was significantly lower than that of the vehicle controls (both p<0.05; Fig. 4B).
6-AN did not affect glycolytic rate and capacity under normoxia (both p>0.05; Fig. 4C) but reduced basal (p<0.01) and maximal respiration (p<0.05; Fig. 4D). No significant differences in glycolytic rate or capacity (both p>0.05; Fig. 4E) or in basal or maximal respiration (p>0.05, Fig. 4F) were seen with 6-AN under hypoxic conditions.
Inhibition of pyruvate oxidation increased cell stemness under normoxia and promotion of pyruvate oxidation reversed the effect of hypoxia in maintaining cell stemness
The glycolytic product, pyruvate, is transported into the mitochondrion by the mitochondrial pyruvate carrier (MPC) for further oxidation. UK5099 is a potent inhibitor of MPC (20). Under normoxic conditions, 20 uM UK5099 promoted cell proliferation (p < 0.01; Fig. 5B) and, although colony formation was unaffected (p>0.05; Fig. 5C), Nanog, Oct4, Klf4 and Sox2, were all upregulated (all p<0.05; Fig. 5D). Moreover, glycolytic rate (p < 0.05) and capacity (p < 0.001) were both higher in the presence of UK5099 (Fig. 5E) and basal (p<0.05) and maximal respiration rates (p < 0.01) were both lower (Fig. 5F). By contrast, UK5099 had opposing effects under hypoxic conditions and suppressed EPC proliferation (Supp. Fig. 1A), producing no effect on either colony formation (P>0.05, Supp. Fig. 1B) nor on expression of Nanog, Klf4 and Sox2. Expression of Oct4 was found to decrease in the presence of UK5099 (Supp. Fig. 1C). Moreover, UK5099 treatment reduced glycolytic capacity (p<0.05; Supp. Fig. 1D) and basal respiration (p<0.05; Supp. Fig. 1E). In combination, the above data suggest that inhibition of pyruvate oxidation promotes glycolysis, reduces mitochondrial respiration and increases EPC stemness under normoxia but has no further benefits for maintenance of stemness under hypoxia.
Pyruvate dehydrogenase (PDH) catalyzes the irreversible oxidative decarboxylation of the glycolytic product, pyruvate, to the TCA cycle substrate, acetyl coenzyme A and flux through PDH, is negatively regulated by pyruvate dehydrogenase kinase (PDK) enzymes which may be inhibited by the compound, AZD7545 (Fig. 5A)(21, 22). Inhibition of PDK1, PDK2 and PDK3 by AZD7545, with the resulting stimulation of pyruvate oxidation (23, 24), suppressed cell proliferation (Fig. 5G), reduced colony formation (p<0.01; Fig. 5H) and reduced expression of Nanog, Oct4, Klf4 and Sox2 (Fig. 5I) under hypoxic conditions. Glycolytic rate and capacity decreased (both p< 0.01, Fig. 5J) and basal and maximal respiration increased (both p < 0.05; Fig. 5K) under the same conditions. Use of AZD7545 under normoxic conditions produced the opposite results. AZD7545 promoted EPC proliferation (Fig. S1F) while colony formation was unaffected (p>0.05; Fig. S1G). Expressions of Nanog (p<0.01), Oct4 (p<0.05) and Klf4 (p<0.01) were all increased (Fig. S1H). In addition, glycolytic rate and capacity increased (both p<0.01; Fig. S1I) while basal and maximal respiration decreased (both p<0.05; Fig. S1J). Overall, it appears that the promotion of pyruvate oxidation resulting from use of AZD7545 reversed the effects of hypoxia in maintaining stemness. Use of AZD7545 did not appear to promote the oxidation of pyruvate in EPCs under normoxic conditions.
Inhibition of the TCA cycle increased cell stemness under normoxia
Citrate synthase (CS), isocitrate dehydrogenase 3 (IDH3), and α-ketoglutarate dehydrogenase (KGDHC) all catalyze irreversible steps in the TCA cycle. Palmitoyl-coenzyme A (PCA) inhibits CS by interacting with the enzyme’s small domain (25, 26). Succinyl phosphonate trisodium salt (SP) inhibit KGDHC isoforms from muscle, bacteria, brain and cultured human fibroblasts (Fig. 6A) (27, 28). No chemical inhibitor of IDH3 was found.
TCA cycle inhibition by PCA promoted cell proliferation under conditions of normoxia (Fig. 6B), although colony formation was unaffected (p>0.05, Fig. 6C). Expression of Nanog, Oct4, Klf4 and Sox2 was upregulated when PCA was present (all p<0.05; Fig. 6D). Moreover, use of PCA increased glycolytic rate and capacity (both p < 0.05; Fig. 6E) and decreased basal (p<0.05) and maximal respiration (p < 0.01; Fig. 6F) compared with controls. Very similar results were obtained with SP (Fig. 6G - 6K). Thus, inhibition of the TCA cycle increases flux through glycolysis and stemness under normoxic conditions.
By contrast, use of PCA or SP had little effect on cell proliferation (all p>0.05; Fig. S2A, 2F), colony formation (all p>0.05; Fig. S2B, 2G), expression of stemness markers (Fig. S2C; 2H), glycolytic rate and capacity (all p>0.05; Fig. S2D, 2I), basal or maximal respiration (all p>0.05; Fig. S2E, 2J) under hypoxic conditions. The implication from these data is that the TCA cycle may already be maximally suppressed under hypoxic conditions and further inhibition has little detectable effect.
Inhibition of the ETC
The complex III inhibitor, antimycin A (AA) was used to block the ETC (29). Use of AA promoted EPC proliferation (Fig. 7A) under normoxic conditions, although there was a significant reduction in colony formation (p<0.05; Fig. 7B). Production of Oct4 and Klf4 mRNA (both p<0.05; Fig. 7C) and glycolytic rate (p<0.05; Fig. 7D) were all stimulated and maximal respiration (p < 0.001; Fig. 7E) reduced compared with controls. Use of AA under hypoxic conditions produced quite different results. Cell proliferation was suppressed (Fig. S3A), colony formation inhibited (p<0.05; Fig. S3B), Oct4 expression decreased (p<0.01; Fig. S3C) and glycolytic capacity reduced (p<0.05; Fig. S3D). Maximal respiration capacity was increased when AA was present under hypoxic conditions (p < 0.05; Fig. S3E).
Adenosine triphosphate (ATP) production
Although a significant suppression of mitochondrial ATP production under-hypoxia (P < 0.001) was found, total cellular ATP production (p>0.05) was unaffected, compared with normoxia (Fig. 8A). Pharmacological manipulation of ATP level was then investigated, although inhibitors of glycolysis or the PPP could not be tested, since they had been shown to suppress EPC growth. UK5099 reduced mitochondrial ATP production (p<0.05) but did not affect total cellular ATP either under normoxia or hypoxia (p>0.05, Fig. 8B). AZD7545 reduced mitochondrial ATP under noxmoxia (p<0.05) but did not affect cellular ATP either under normoxia or hypoxia (both p>0.05; Fig. 8C). Similarly, the two TCA cycle inhibitors, PCA and SP, reduced mitochondrial ATP under normoxia (all p<0.05) but did not affect cellular ATP level either under normoxia or hypoxia (all p>0.05; Fig. 8D-E). Use of AA also reduced mitochondrial ATP level under both normoxic (p<0.001) and hypoxic (p<0.05) conditions but had no effect on total cellular ATP level under either condition (p>0.05; Fig. 8F)