Blockade of MCU-Mediated Ca ²⁺ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation

Mitochondrial Ca ²⁺ uniporter (MCU)-mediated Ca ²⁺ uptake promotes the buildup of reducing equivalents that fuel oxidative phosphorylation for cellular metabolism. Although MCU modulates mitochondrial bioenergetics, its function in energy homeostasis in vivo remains elusive. Here we demonstrate that deletion of the Mcu gene in mouse liver (MCU ^Δhep ) and in Danio rerio by CRISPR/Cas9 inhibits mitochondrial Ca ²⁺ ( _m Ca ²⁺ ) uptake, delays cytosolic Ca ²⁺ ( _c Ca ²⁺ ) clearance, reduces oxidative phosphorylation, and leads to increased lipid accumulation. Elevated hepatic lipids in MCU ^Δhep were a direct result of extramitochondrial Ca ²⁺ -dependent protein phosphatase-4 (PP4) activity, which dephosphorylates AMPK. Loss of AMPK recapitulates hepatic lipid accumulation without changes in MCU-mediated Ca ²⁺ uptake. Furthermore, reconstitution of active AMPK, or PP4 knockdown, enhances lipid clearance in MCU ^Δhep hepatocytes. Conversely, gain-of-function MCU promotes rapid _m Ca ²⁺ uptake, decreases PP4 levels, and reduces hepatic lipid accumulation. Thus, our work uncovers an MCU/PP4/AMPK molecular cascade that links Ca ²⁺ dynamics to hepatic lipid metabolism. Hepatic mitochondrial Ca ²⁺ shapes bioenergetics and lipid homeostasis. Tomar et al. demonstrate that MCU-mediated _c Ca ²⁺ buffering serves as a crucial step in controlling hepatic fuel metabolism through an MCU/PP4/AMPK molecular cascade. Identification of these molecular signaling events aids in understanding how perturbation of mitochondrial ion homeostasis may contribute to the etiology of metabolic disorders.