Metabolic preconditioning of mammalian cells: mimetic agents for hypoxia lack fidelity in promoting phosphorylation of pyruvate dehydrogenase

Abstract

Induction of HIF-1α by oxygen limitation promotes increased phosphorylation and catalytic depression of mitochondrial pyruvate dehydrogenase (PDH) and an enhanced glycolytic poise in cells. Cobalt chloride and desferrioxamine are widely used as mimics for hypoxia because they increase the levels of HIF-1α. We evaluated the ability of these agents to elicit selected physiological responses to hypoxia as a means to metabolically precondition mammalian cells, but without the detrimental effects of hypoxia. We show that, while CoCl(2) does increase HIF-1α in a dose-dependent manner, it unexpectedly and strikingly decreases PDH phosphorylation at E1α sites 1, 2, and 3 (Ser(293), Ser(300), and Ser(232), respectively) in HepG2 cells. This same effect is also observed for site 1 in mouse NIH/3T3 fibroblasts and J774 macrophages. CoCl(2) unexpectedly decreases the mRNA expression for PDH kinase-2 in HepG2 cells, which likely explains the dephosphorylation of PDH observed. And nor does desferrioxamine promote the expected increase in PDH phosphorylation. Dimethyloxaloylglycine (a prolyl hydroxylase inhibitor) performs better in this regard, but failed to promote the stronger effects seen with hypoxia. Consequently, CoCl(2) and desferrioxamine are unreliable mimics of hypoxia for physiological events downstream of HIF-1α stabilization. Our study demonstrates that mimetic chemicals must be chosen with caution and evaluated thoroughly if bona fide cellular outcomes are to be promoted with fidelity.

Fig. 1

Western blot analysis of HepG2 cells that were incubated with the indicated concentrations of CoCl₂ or exposed to hypoxia (“H”; 1% O₂) for 24 h. Amounts of HIF-1α, the phosphorylation state of PDH site 1 (E1α subunit, phosphoserine²⁹³), site 2 (E1α subunit, phosphoserine³⁰⁰), site 3 (E1α subunit, phosphoserine²³²), and the β-actin loading control are shown.

Fig. 2

a Western blot analysis of the effect of 100 μM CoCl₂ on HepG2 cells across the indicated time course. Amounts of HIF-1α, the phosphorylation state of PDH site 1 (E1α subunit, phosphoserine²⁹³) and the β-actin loading control are shown. b Western blot analysis of NIH/3T3 fibroblasts that were incubated with the indicated concentrations of CoCl₂ or exposed to hypoxia (“H”; 1% O₂) for 24 h. The phosphorylation state of PDH site 1 (E1α subunit, phosphoserine²⁹³) and β-actin loading control are indicated. c Western blot analysis of J774 macrophages that were incubated with the indicated concentrations of CoCl₂ or exposed to hypoxia (“H”; 1% O₂) for 24 h. The phosphorylation state of PDH site 1 (E1α subunit, phosphoserine²⁹³) and β-actin loading control are shown.

Fig. 3

a Western blot analysis of HepG2 cells that were incubated with the indicated concentrations of desferrioxamine or exposed to hypoxia (“H”; 1% O₂) for 24 h. Amounts of HIF-1α, the phosphorylation state of PDH site 1 (E1α subunit, phosphoserine²⁹³), site 2 (E1α subunit, phosphoserine³⁰⁰), site 3 (E1α subunit, phosphoserine²³²) and the β-actin loading control are documented. b Western blot analysis of HepG2 cells that were incubated with the indicated concentrations of DMOG or exposed to hypoxia (“H”; 1% O₂) for 24 h. Amounts of HIF-1α, the phosphorylation state of PDH site 1 (E1α subunit, phosphoserine²⁹³), site 2 (E1α subunit, phosphoserine³⁰⁰), site 3 (E1α subunit, phosphoserine²³²) and the β-actin loading control are shown.

Fig. 4

Quantitative impact of 24 h incubations of HepG2 cells with CoCl₂ (solid lines) and desferrioxamine (dashed lines) on the phosphorylation states of PDH site 1 (closed circles), site 2 (closed squares) and site 3 (closed triangles). All values are normalized to β-actin. Slopes of the linear regressions for sites 1 and 3 were not significantly different from zero (p > 0.05) for the HepG2 cells incubated with desferrioxamine, while the small positive slope for site 2 was significantly different from zero (p = 0.013). Inset: Elevation of HIF-1α (normalized to β-actin) in HepG2 cells after 24 h exposures to CoCl₂ (open circles) and desferrioxamine (open squares).

Fig. 5

Quantification of Western blots illustrates the impact of 24 h incubation with DMOG on the phosphorylation states of PDH site 1 (circles), site 2 (squares) and site 3 (triangles) in HepG2 cells. All values are normalized to β-actin. Inset: Elevation of HIF-1α (normalized to β-actin) in HepG2 cells after 24 h exposures to DMOG.

Fig. 6

Expression of PDK-2 mRNA in HepG2 cells that were incubated with the indicated concentrations of CoCl₂ or exposed to hypoxia (“H”; 1% O₂) for 24 h. a PCR analyses of mRNA for PDK-2 and β-actin (loading control) are shown. b Results obtained by rtqPCR. PDK-2 mRNA was normalized to a housekeeper gene (β-actin mRNA) and expressed as a percentage of the untreated/normoxic control. Asterisks indicate significant differences from the hypoxia treatment (“H”) (one-way ANOVA, n = 9, mean ± 1 SD; p ≤ 0.05).