Metabolic and oncogenic adaptations to pyruvate dehydrogenase inactivation in fibroblasts

Huabo Wang,Jie Lu,Sucheta Kulkarni,Weiqi Zhang,Joanna E Gorka,Jordan A Mandel,Eric S Goetzman,Edward V Prochownik

doi:10.1074/jbc.ra118.005200

Abstract

Eukaryotic cell metabolism consists of processes that generate available energy, such as glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (Oxphos), and those that consume it, including macromolecular synthesis, the maintenance of ionic gradients, and cellular detoxification. By converting pyruvate to acetyl-CoA (AcCoA), the pyruvate dehydrogenase (PDH) complex (PDC) links glycolysis and the TCA cycle. Surprisingly, disrupting the connection between glycolysis and the TCA cycle by inactivation of PDC has only minor effects on cell replication. However, the molecular basis for this metabolic re-equilibration is unclear. We report here that CRISPR/Cas9-generated PDH-knockout (PDH-KO) rat fibroblasts reprogrammed their metabolism and their response to short-term c-Myc (Myc) oncoprotein overexpression. PDH-KO cells replicated normally but produced surprisingly little lactate. They also exhibited higher rates of glycolysis and Oxphos. In addition, PDH-KO cells showed altered cytoplasmic and mitochondrial pH, redox states, and mitochondrial membrane potential (ΔΨM). Conditionally activated Myc expression affected some of these parameters in a PDH-dependent manner. PDH-KO cells had increased oxygen consumption rates in response to glutamate, but not to malate, and were depleted in all TCA cycle substrates between α-ketoglutarate and malate despite high rates of glutaminolysis, as determined by flux studies with isotopically labeled glutamine. Malate and pyruvate were diverted to produce aspartate, thereby potentially explaining the failure to accumulate lactate. We conclude that PDH-KO cells maintain proliferative capacity by utilizing glutamine to supply high rates of AcCoA-independent flux through the bottom portion of the TCA cycle while accumulating pyruvate and aspartate that rescue their redox defects.

Highlights

Eukaryotic cell metabolism consists of processes that generate available energy, such as glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (Oxphos), and those that consume it, including macromolecular synthesis, the maintenance of ionic gradients, and cellular detoxification
Because PDK1 and PDP2 are themselves regulated by metabolites such as ATP, ADP, NADH, and AcCoA [6, 15], it seems likely that the loss of pyruvate dehydrogenase (PDH)␣1 phosphorylation in WT cells in response to MycER induction is more dependent on these enzymes’ activities than on their actual levels as occurs in hepatocytes [9, 13, 14]
Unlike WT cells, which rapidly increased NADH levels in response to MycER activation, KO cells showed no such response, both cell types decreased their NADϩ/NADH ratios in response to Myc activation (Fig. 1F). These findings suggested that the increased NADH content of WT cells in response to MycER activation originated with the PDH reaction, which generates NADH as a by-product of the intra-mitochondrial oxidation of pyruvate to AcCoA

Summary

To whom correspondence should be addressed

Answers to such questions could explain how cells normally achieve metabolic re-equilibration to accommodate nutritional and neoplastic challenges We address these and additional questions by exploring the consequences of pdha inactivation in rat fibroblasts in which the c-Myc (Myc) oncoprotein, fused to the hormone-binding domain of the estrogen receptor, can be conditionally activated by 4-hydroxytamoxifen (4OHT) [11, 12]. We demonstrate that these so-called Rat1a–MycER cells undergo significant metabolic re-programming that compensates for the loss of PDC activity, restores normal levels of AcCoA, and allows them to proliferate as well as their wildtype (WT) counterparts. These findings point to major similarities and differences in the ways that hepatocytes and fibroblasts cope with the loss of PDH and emphasize the flexibility that can be marshaled in response to what should be a devastating metabolic deficit

Results

Discussion

Experimental procedures