Elevated Exogenous Pyruvate Potentiates Mesodermal Differentiation through Metabolic Modulation and AMPK/mTOR Pathway in Human Embryonic Stem Cells

摘要

class="head no_bottom_margin" id="sec1title">IntroductionBalanced cellular metabolism is essential for cell survival and proliferation in embryogenesis and cancer biology. Metabolic balance is shifted when the cell fate is changed in somatic reprogramming or stem cell differentiation (, , ). Increasing evidence shows that the modulation of cellular metabolism affects self-renewal and cell-fate determination (, , ). Pyruvate is the keystone molecule in mammalian metabolism and has been widely used to culture human preimplantation embryos and human embryonic stem cells (hESCs) (, , ). Here we investigated the impact of exogenous pyruvate on hESC metabolism, and further revealed its roles in signal transduction and cell-fate determination.Pyruvate is the main product from glycolysis, and can be utilized in either lactate production or generation of acetyl-coenzyme A (CoA) for the tricarboxylic acid (TCA) cycle (). In pluripotent stem cells, pyruvate is mainly converted to lactic acid via lactic acid fermentation, and the rest is utilized through the TCA cycle to produce energy in oxidative phosphorylation (, , ). In contrast, pyruvate is mainly metabolized through the TCA cycle in somatic cells (). The interference of pyruvate-related metabolism has a profound impact on autophagy, cell survival, and cell differentiation in stem cells (, href="#bib32" rid="bib32" class=" bibr popnode">Moussaieff et al., 2015b, href="#bib33" rid="bib33" class=" bibr popnode">Nagaraj et al., 2017). For example, the inhibition of glycolysis suppresses endogenous pyruvate production and induces differentiation (href="#bib3" rid="bib3" class=" bibr popnode">Burgess et al., 2014, href="#bib27" rid="bib27" class=" bibr popnode">Lunt and Vander Heiden, 2011, href="#bib32" rid="bib32" class=" bibr popnode">Moussaieff et al., 2015b, href="#bib49" rid="bib49" class=" bibr popnode">Vander Heiden et al., 2009). During gastrulation and stem cell differentiation, the balance of pyruvate usage is shifted from glycolysis to mitochondrial oxidative metabolism (href="#bib4" rid="bib4" class=" bibr popnode">Butcher et al., 1998, href="#bib12" rid="bib12" class=" bibr popnode">Conaghan et al., 1993, href="#bib20" rid="bib20" class=" bibr popnode">International Stem Cell Initiative et al., 2010). All the evidence suggests that pyruvate plays a central role in stem cell regulation.Cells in our body are constantly under the influence of extracellular pyruvate, which can be imported through its transporters for intracellular metabolism (href="#bib18" rid="bib18" class=" bibr popnode">Halestrap et al., 1990, href="#bib38" rid="bib38" class=" bibr popnode">Poole and Halestrap, 1993). Exogenous pyruvate has been widely used in clinical practice and basic research. In clinical practice, systemic administration of pyruvate is used as a therapeutic intervention for cardiac, neurological, and acid-base disorders (href="#bib42" rid="bib42" class=" bibr popnode">Salahudeen et al., 1991). In basic research, up to 40 mM pyruvate has been used in cell culture, and many basic culture media contain 0.5–1 mM pyruvate (href="#bib8" rid="bib8" class=" bibr popnode">Chavez-Perez et al., 2011, href="#bib19" rid="bib19" class=" bibr popnode">Hereng et al., 2011, href="#bib51" rid="bib51" class=" bibr popnode">Watanabe et al., 2017). Pyruvate is used in all preimplantation embryo and hESC media (href="#bib4" rid="bib4" class=" bibr popnode">Butcher et al., 1998, href="#bib12" rid="bib12" class=" bibr popnode">Conaghan et al., 1993, href="#bib20" rid="bib20" class=" bibr popnode">International Stem Cell Initiative et al., 2010), and displays cellular protection to stem cells (href="#bib14" rid="bib14" class=" bibr popnode">Gonzalez et al., 2005, href="#bib19" rid="bib19" class=" bibr popnode">Hereng et al., 2011, href="#bib39" rid="bib39" class=" bibr popnode">Ramos-Ibeas et al., 2017, href="#bib42" rid="bib42" class=" bibr popnode">Salahudeen et al., 1991). In our laboratory we use E8 medium, which contains 0.5 mM pyruvate, to maintain hESCs.Exogenous pyruvate is important in early embryogenesis. Pyruvate uptake from media is essential for preimplantation embryos (href="#bib4" rid="bib4" class=" bibr popnode">Butcher et al., 1998). Pyruvate regulates the nuclear localization of TCA-cycle enzymes and is required for the genomic activation in mammalian zygote (href="#bib33" rid="bib33" class=" bibr popnode">Nagaraj et al., 2017). At the same time, pyruvate also plays a role in epigenetic modification. Pyruvate is converted to acetyl-CoA, which contributes to histone acetylation (href="#bib32" rid="bib32" class=" bibr popnode">Moussaieff et al., 2015b, href="#bib37" rid="bib37" class=" bibr popnode">Pietrocola et al., 2015). Elevated exogenous pyruvate also promotes the nuclear translocation of pyruvate dehydrogenase (PDH) for histone acetylation (href="#bib46" rid="bib46" class=" bibr popnode">Sutendra et al., 2014). In addition, pyruvate generates α-ketoglutarate (α-KG) in the TCA cycle, which is involved in DNA methylation (href="#bib7" rid="bib7" class=" bibr popnode">Carey et al., 2015, href="#bib32" rid="bib32" class=" bibr popnode">Moussaieff et al., 2015b, href="#bib47" rid="bib47" class=" bibr popnode">TeSlaa et al., 2016). Given its essential roles in embryogenesis, it would be important to explore the regulatory functions of pyruvate in hESC maintenance and differentiation.Pluripotency and differentiation are influenced by exogenous metabolites such as methionine, serine, acetate, and membrane-permeable α-KG analog (dimethyl α-KG, DM2OG) (href="#bib7" rid="bib7" class=" bibr popnode">Carey et al., 2015, href="#bib23" rid="bib23" class=" bibr popnode">Kilberg et al., 2016, href="#bib33" rid="bib33" class=" bibr popnode">Nagaraj et al., 2017, href="#bib44" rid="bib44" class=" bibr popnode">Shiraki et al., 2014, href="#bib50" rid="bib50" class=" bibr popnode">Wang et al., 2009). We and others have shown that some exogenous metabolites can also influence signaling cascades (href="#bib30" rid="bib30" class=" bibr popnode">Meng et al., 2018b, href="#bib6" rid="bib6" class=" bibr popnode">Carcamo et al., 2004). Because of pyruvate's critical roles in metabolism and early embryogenesis, we hypothesize that exogenous pyruvate can influence metabolism and signaling pathways in hESCs. In this report, we demonstrate that elevated exogenous pyruvate alters the metabolic profile and enhances oxidative metabolism. Pyruvate changes the expression of metabolic genes and the phosphorylation of metabolic enzymes. In differentiation, pyruvate potentiates mesodermal and endodermal lineage-specific differentiation. We further show that pyruvate stimulates mesoderm differentiation by modulating AMP kinase (AMPK) and mammalian target of rapamycin (mTOR) pathways. Our study highlights pyruvate as a key modulator that integrates metabolism and signal transduction in stem cell regulation.