A cell culture-based method for interrogating muscle to liver communication via secreted proteins.

Inter-organ communication, including the release of secreted proteins, plays a key role in synchronized physiological responses and organismal homeostasis. Recent studies have emphasized functions of muscle-secreted proteins (i.e., myokines), in regulating metabolic pathways and improving metabolic dysfunction distally in the liver. Thus, experimental workflows to study myokines and their impact on target cell types are of scientific value. Here, we describe a cell culture-based method to investigate communication from muscle to liver mediated by secreted proteins. Briefly, C2C12 myoblasts are differentiated into myotubes, myotube-conditioned media is collected, and myotube-secreted proteins are isolated and stored. To demonstrate the utility of this method, AML12 hepatocytes were treated with myotube-secreted proteins and effects on bioenergetics were assessed. This method can be useful as a proof of principle tool, for mechanistic studies, or paired with proteomic or biochemical analyses to identify novel myokines. We also envision it is adaptable in terms of cell type, downstream application, and signaling direction.