Cells are incredibly complex entities that capture the biological contents of the intact organism. Approximately 20,000 canonical, unmodified proteins make up the human proteome, but through alternative splicing, single amino acid polymorphisms, and post-translational modifications, the number of distinct proteins may be 100-fold higher. The number of protein-coding genes is similar, but if one includes noncoding regulatory and enzymatic genes, the number explodes to close to 50,000. More than a decade of intense investigation has led to estimates for the numbers of identified human metabolites, relevant biological pathways, and metabolite–disease interactions at more than 350,000, 25,000, and 5,500, respectively.
Clearly, quantifying and characterizing even a small fraction of molecules and pathways relevant to disease is impossible. Instead of measuring and identifying what cells contain, one can often learn as much about molecules and pathways by studying the bioenergetics that drive a cell’s formation and activity—in other words, by analyzing what cells do instead of what they contain.
Recommended eBook
Download this ebook now and learn more about how new workflows combining functional cellular and metabolism analysis with imaging and fluorescence detection capabilities are delivering better solutions for a wide variety of research applications.Cells build and maintain their complex pathways by managing their energy flux. Energy management—its allocation to growth, survival, and reproduction—could arguably be called cells’ most critical function. Real-time measurements of cellular bioenergetics therefore provide a clear window into the critical functions driving cell signaling, proliferation, activation, toxicity, and biosynthesis, both in normal cells and in disease states.
Cellular bioenergetics, the study of how cells use and process energy—typically by making and breaking chemical bonds in adenosine triphosphate (ATP)—is the underlying mechanism for cellular respiration and photosynthesis. Examples of bioenergetic processes include glycolysis, gluconeogenesis, the citric acid cycle, ketosis, and oxidative phosphorylation.
The study of bioenergetics is possible because, despite their great complexity and interconnectedness, energy-processing systems in cells follow the laws of thermodynamics. The first law, which states that cellular energy is neither created nor destroyed but may change form (e.g., from chemical to mechanical), allows for “bookkeeping” of inputs and production of Gibbs free energy (ΔG = ΔH – TΔS). The second law states that the entropy of the universe increases, even as cells are continually fighting to maintain order within their boundaries.
Measuring metabolic flux
Metabolic fluxes were traditionally measured through radiolabeling—for example, by measuring accumulation of tritiated water from the metabolism of 3-3H-D-glucose. Radiolabeling can be quite specific but lack precision, be labor and time intensive, and require large quantities of sample.
Another approach uses microphysiometric sensors—for example, Clark-type electrodes that measure oxygen consumption rate (OCR) or probe for extracellular acidification rate (ECAR). Oxygen consumption is a proxy for mitochondrial respiration, while acid efflux quantifies the lactic acid formed during glycolysis. Researchers from Seahorse Bioscience and the University of South Carolina first reported combining OCR and ECAR measurements within a single platform, a technique that “enables a more comprehensive assessment of cellular energetics and the ability to determine the relative contribution of these two dominant energy yielding pathways.”
Conclusion
Cellular bioenergetics, the key to all cellular activity, holds promise for advancing every aspect of cell-based research. Numerous papers have appeared describing bioenergetics as the key to understanding cell-based therapies, cancer research, immunology, virology, drug discovery, stem cell research, and abnormal metabolism. All such studies involve real-time assessments of cellular function, phenotype, and fate.
Research applications
Immunotherapy: Developing immune cell therapies requires an in-depth understanding of immune cell function. This requires key technologies from high-efficiency gene editing to assessment of real-time cell function, phenotype, and fate. New tools can help reseachers develop potent and safe therapeutics.
Cancer research: Cancer cells reprogram their metabolism in order to generate the energy and building blocks they need to proliferate. Now, researchers have a window into the Warburg effect, fuel usage, and other events that drive tumor cell biology.
Immunology research: Immunometabolism has emerged as a crucial component to understand the connection between metabolic pathways and immune responses. Novel technology enables researchers to understand and perturb immune activation and response in real time through metabolic measurements.
Drug discovery: From target identification and validation to toxicology and mechanism of action, being able to collect direct measurements of cell and energy metabolism will allow you to expand your drug discovery pipeline by targeting metabolic pathways, assessing compounds for mitochondrial toxicity, and better understanding their mechanisms.
Stem cell research: Metabolic shift in induced pluripotent stem cells (iPSCs) has a significant importance in establishing pluripotent identity. Now, researchers can predict the ability of somatic cells to reprogram themselves into iPSCs and predict how PSCs alter their metabolism during the differentiation process to terminally differentiated cells.
Metabolic disorders: Research into metabolic profiles and changes is enabling insight into browning, substrate and nutrient utilization, and inflammation. With new tools, researchers have the capability to examine functional metabolism in models of cellular metabolic syndromes.
To learn more about the tools and technologies that are making it possible to measure cellular bioenergetics in real time, download our free eBook now.