Pharmacologic Reprogramming Designed to Induce a Warburg Effect in Porcine Fetal Fibroblasts Alters Gene Expression and Quantities of Metabolites from Conditioned Media Without Increased Cell Proliferation

Abstract

The Warburg effect is a metabolic phenomenon characterized by increased glycolytic activity, decreased mitochondrial oxidative phosphorylation, and the production of lactate. This metabolic phenotype is characterized in rapidly proliferative cell types such as cancerous cells and embryonic stem cells. We hypothesized that a Warburg-like metabolism could be achieved in other cell types by treatment with pharmacological agents, which might, in turn, facilitate nuclear reprogramming. The aim of this study was to treat fibroblasts with CPI-613 and PS48 to induce a Warburg-like metabolic state. We demonstrate that treatment with both drugs altered the expression of 69 genes and changed the level of 21 metabolites in conditioned culture media, but did not induce higher proliferation compared to the control treatment. These results support a role for the reverse Warburg effect, whereby cancer cells induce cancer-associated fibroblast cells in the surrounding stroma to exhibit the metabolically characterized Warburg effect. Cancer-associated fibroblasts then produce and secrete metabolites such as pyruvate to supply the cancerous cells, thereby supporting tumor growth and metastasis. While anticipating an increase in the production of lactate and increased cellular proliferation, both hallmarks of the Warburg effect, we instead observed increased secretion of pyruvate without changes in proliferation.

Pharmacologic treatment of donor cells induced to have a Warburg effect‐like metabolism does not alter embryonic development in vitro or survival during early gestation when used in somatic cell nuclear transfer in pigs

“…Our objective was to determine if a drug‐induced Warburg‐like effect in donor cells prior to SCNT would improve clone survival, however we also tested CPI and PS48 in embryo culture media. Selected dosages of CPI (100 μM) and PS48 (10 μM) were based on prior research demonstrating a dosage‐dependent loss of mitochondrial membrane potential in the same porcine fetal fibroblast line which was used for the in vitro experiments in this study (Mordhorst et al, ). The mixture of these drugs at these concentrations induced changes in expression of phosphoenolpyruvate carboxykinase 2 , phosphoglycerate dehydrogenase , phosphoserine phosphatase as well as the amount of pyruvate, fructose, serine, and glutamine in conditioned media indicating changes in metabolic pathways including glycolysis and serine biosynthesis (Mordhorst et al, ).…”

“…Incubators were maintained at 38.5 °C with a humidified atmosphere of 5% oxygen, 5% carbon dioxide, and 90% nitrogen during experiments. Cells were cultured as per previously established protocol (Mordhorst et al, ) to maintain similar cell densities across treatments at passaging in an effort to keep drug exposure amongst treatments comparable. Using DMSO for suspension, CPI stocks were diluted to 100 mM and PS48 stocks were diluted to 10 mM to eliminate the confounding of DMSO concentration with pharmaceutical treatment.…”

“…Incubators were maintained at 38.5°C with a humidified atmosphere of 5% oxygen, 5% carbon dioxide, and 90% nitrogen during experiments. Cells were cultured as per previously established protocol(Mordhorst et al, 2018) to maintain similar cell densities across treatments at passaging in an effort to keep drug exposure…”

Porcine Fetal-Derived Fibroblasts Alter Gene Expression and Mitochondria to Compensate for Hypoxic Stress During Culture

“…The current study is novel in its approach to program metabolism of donor fibroblasts to a Warburg effect-like state similar to that of preimplantation embryos in an effort to improve somatic cell nuclear transfer. To date, we do not know of any other groups which have tried similar strategies outside of our laboratory (Mordhorst et al, 2018a , 2018b ).…”

Applications of omics and nanotechnology to improve pig embryo production in vitro

“…Another candidate was CPI‐613 (6, 8‐bis(benzylthio)octanoic acid) to inhibit pyruvate dehydrogenase and α‐ketoglutarate dehydrogenase, resulting in decreased mitochondrial metabolism. Treatment with the compounds did not promote proliferation of the donor cells but increased secretion of pyruvate into the medium which is a characteristic of the reverse WE (Mordhorst, Murphy, Ross, et al, ; Pavlides et al, ). Furthermore, donor cells exposed to hypoxic culture conditions (1.25% O 2 ) demonstrated increased abundance of transcripts involved in glycolysis and smaller mitochondria compared to control cells cultured at 5% O 2 , indicating that this method may be effective for inducing the WE (Mordhorst, Murphy, Schauflinger, et al, ).…”