Metabolic and oncogenic adaptations to pyruvate dehydrogenase inactivation in fibroblasts

Abstract

Edited by Eric R. FearonEukaryotic 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 (⌬⌿M). 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 ␣-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.

Reprogramming of aerobic glycolysis in non‐transformed mouse liver with pyruvate dehydrogenase complex deficiency

“…HIF‐1α also decreases the transport of pyruvate (Eboli et al, 1977; Paradies et al, 1983; Schell et al, 2014) and its oxidation by PDC in the mitochondria, causing reduced oxidative respiration (Battello et al, 2016). In cancer cells and rapidly growing nontransformed cells with enhanced aerobic glycolysis, reduced pyruvate oxidation by PDC is achieved by at least five complimentary mechanisms: (a) posttranslational phosphorylation of specific α subunit tyrosine residues of PDH by oncogenic kinases [such as fibroblast growth factor receptor 1 (FGFR1)] causing inhibition of PDC activity (Fan et al, 2014; Hitosugi et al, 2011), (b) a novel posttranslational modification of pyruvate dehydrogenase phosphatase 1 (PDP1) by tyrosine phosphorylation by oncogene kinases to inhibit its activity (Shan et al, 2014), (c) increased transcription of PDK1 to increase PDK1 activity to exert greater inhibition of PDH via serine phosphorylation of the α subunit, and (d) decreased levels of PDH and PDP2 (Jackson et al, 2017; Wang et al, 2019), and (e) the modulation of both PDK1 and PDP2 by small molecule products of oxidative phosphorylation such as ATP, NADH and acetyl‐CoA (Roche et al, 2001). The effects in (a) and (b) are exerted by oncogene kinases, and in (c) by HIF‐1α.…”

“…The effects in (a) and (b) are exerted by oncogene kinases, and in (c) by HIF‐1α. These modulators, however, also regulate many other key genes or proteins responsible for increased aerobic glycolysis and oxidative phosphorylation (Wang et al, 2019). The findings presented here (Figure 7) show that the elimination of PDC activity in otherwise normal mouse liver by Pdha1 gene deletion (without involving any cancer‐mediated modulators described above) is sufficient to induce Warburg‐type respiration as a result of PDC deficiency (Jackson et al, 2017; Reznik et al, 2017).…”

“…In a recent study, compared to wild‐type rat fibroblasts, CRISPR/Cas9‐mediated Pdha1 ‐knockout rat fibroblasts showed a modest increase in glucose uptake with increased steady‐state levels of several glycolytic intermediates, notably pyruvate being very high without any significant change in lactate level under normal culture conditions (Wang et al, 2019). Interestingly, the levels of fructose and mannose‐6‐phosphate were also increased in PDH‐knockout rat fibroblasts, most likely derived from the selective glycolytic intermediates (Wang et al, 2019). PDH‐knockout rat fibroblasts showed lower oxygen consumption rates, indicating a greater dependence on glycolysis for ATP generation (Wang et al, 2019).…”

“…Interestingly, the levels of fructose and mannose‐6‐phosphate were also increased in PDH‐knockout rat fibroblasts, most likely derived from the selective glycolytic intermediates (Wang et al, 2019). PDH‐knockout rat fibroblasts showed lower oxygen consumption rates, indicating a greater dependence on glycolysis for ATP generation (Wang et al, 2019). Furthermore, there were significant changes in the steady‐state levels of the tricarboxylic acid cycle intermediates, a significant increase in oxaloacetate and reduction in the levels of the intermediates between α‐ketoglutarate to malate in PDH‐knockout rat fibroblasts compared with wild‐type fibroblasts (Wang et al, 2019).…”

“…These changes not only support increased demand for ATP generation by mitochondrial oxidative phosphorylation for biosynthetic processes for rapidly growing cancers but also provide acetyl‐CoA as the precursor for biosynthesis of lipids such as fatty acids and cholesterol for rapidly proliferating cells (Chen et al, 2018). Additionally, pyruvate carboxylase‐mediated anaplerosis supports survival and proliferation of some cancers and rapidly growing nontransformed fibroblasts (Sellers et al, 2015; Wang et al, 2019).…”