mTORC1 senses nutrients and growth factors and phosphorylates downstream targets, including the transcription factor TFEB, to coordinate metabolic supply and demand. These functions position mTORC1 as a central controller of cellular homeostasis, but the behavior of this system in individual cells has not been well characterized. Here, we provide measurements necessary to refine quantitative models for mTORC1 as a metabolic controller. We developed a series of fluorescent protein-TFEB fusions and a multiplexed immunofluorescence approach to investigate how combinations of stimuli jointly regulate mTORC1 signaling at the single-cell level. Live imaging of individual MCF10A cells confirmed that mTORC1-TFEB signaling responds continuously to individual, sequential, or simultaneous treatment with amino acids and the growth factor insulin. Under physiologically relevant concentrations of amino acids, we observe correlated fluctuations in TFEB, AMPK, and AKT signaling that indicate continuous activity adjustments to nutrient availability. Using partial least squares regression modeling, we show that these continuous gradations are connected to protein synthesis rate via a distributed network of mTORC1 effectors, providing quantitative support for the qualitative model of mTORC1 as a homeostatic controller and clarifying its functional behavior within individual cells.

Insufficient insulin secretion to meet metabolic demand results in diabetes. The intracellular flux of Ca²⁺ into β-cells triggers insulin release. Since genetics strongly influences variation in islet secretory responses, we surveyed islet Ca²⁺ dynamics in eight genetically diverse mouse strains. We found high strain variation in response to four conditions: (1) 8 mM glucose; (2) 8 mM glucose plus amino acids; (3) 8 mM glucose, amino acids, plus 10 nM glucose-dependent insulinotropic polypeptide (GIP); and (4) 2 mM glucose. These stimuli interrogate β-cell function, α- to β-cell signaling, and incretin responses. We then correlated components of the Ca²⁺ waveforms to islet protein abundances in the same strains used for the Ca²⁺ measurements. To focus on proteins relevant to human islet function, we identified human orthologues of correlated mouse proteins that are proximal to glycemic-associated single-nucleotide polymorphisms in human genome-wide association studies. Several orthologues have previously been shown to regulate insulin secretion (e.g. ABCC8, PCSK1, and GCK), supporting our mouse-to-human integration as a discovery platform. By integrating these data, we nominate novel regulators of islet Ca²⁺ oscillations and insulin secretion with potential relevance for human islet function. We also provide a resource for identifying appropriate mouse strains in which to study these regulators.