Radioisotope-Based Protocol for Determination of Central Carbon Metabolism in T Cells.

T lymphocytes are the major components of the adaptive immune system. It's been known that T cells are able to engage a diverse range of metabolic programs to meet the metabolic demands during their life cycle from early development, activation to functional differentiation. Central carbon metabolic pathways provide energy, reducing power, and biosynthetic precursors to support T cell homeostasis, proliferation, and immune functions. As such, quantitative or semiquantitative analysis of central carbon metabolic flux activities offers mechanistic details, as well as insights into the regulation of metabolic pathways and the impact of changing metabolic programs on T cell life cycle. Global profiling of cellular metabolites by mass spectrometry-based metabolomics and metabolic flux analysis (MFA) using radioactive and nonradioactive/stable isotope approaches are powerful tools for determination of central carbon metabolic pathway activity. Here, we describe in detail the procedure for the radioisotope-based approach of analyzing central carbon metabolic fluxes in T cells.

Phosphorylation of CDC25C by AMP-activated protein kinase mediates a metabolic checkpoint during cell-cycle G2/M-phase transition.

From unicellular to multicellular organisms, cell-cycle progression is tightly coupled to biosynthetic and bioenergetic demands. Accumulating evidence has demonstrated the G1/S-phase transition as a key checkpoint where cells respond to their metabolic status and commit to replicating the genome. However, the mechanism underlying the coordination of metabolism and the G2/M-phase transition in mammalian cells remains unclear. Here, we show that the activation of AMP-activated protein kinase (AMPK), a highly conserved cellular energy sensor, significantly delays mitosis entry. The cell-cycle G2/M-phase transition is controlled by mitotic cyclin-dependent kinase complex (CDC2-cyclin B), which is inactivated by WEE1 family protein kinases and activated by the opposing phosphatase CDC25C. AMPK directly phosphorylates CDC25C on serine 216, a well-conserved inhibitory phosphorylation event, which has been shown to mediate DNA damage-induced G2-phase arrest. The acute induction of CDC25C or suppression of WEE1 partially restores mitosis entry in the context of AMPK activation. These findings suggest that AMPK-dependent phosphorylation of CDC25C orchestrates a metabolic checkpoint for the cell-cycle G2/M-phase transition.

MYC and HIF in shaping immune response and immune metabolism.

Upon antigen stimulation, quiescent naive T cells undergo a phase of cell mass accumulation followed by cell cycle entry, clonal expansion, differentiation into functional subsets and back again to a quiescent state as they develop into memory cells. The transitions between these distinct cellular states place unique metabolic demands on energy, redox and biosynthesis. To fulfill these demands, T cells switch back and forth between their primary catabolic pathways. While quiescent naive and memory T cells largely rely on the oxidation of fatty acids and glucose, active T cells rely on glycolysis and glutaminolysis to sustain cell growth, proliferation and differentiation. Beyond several key signaling kinase cascades, the hypoxia inducible factor 1 (HIF-1) and the proto-oncogene MYC, act alone or in concert, to coordinate T cell metabolic reprogramming, cell proliferation, functional differentiation and apoptosis, enabling a robust T cell-mediated adaptive immune response.