Abstract Metabolic diseases include obesity and type 2 diabetes (T2D) are associated with exacerbated health risks that can be life threatening such as heart complications, viral infections, or cancer. Current therapies to treat obesity are based on exercise, diet, and/or bariatric surgery that not always are possible or succeed due to genetic components, non-compliance, or excessive cost. There is a need to understand the mechanisms that sustain energy balance to provide more efficient and cost-effective therapies. Activation of adaptive thermogenesis is an attractive approach to combat obesity/T2D. Increased thermogenic and metabolic function in response to lower temperatures or high calorie diets occurs, at least in part, in specialized fat cells, brown and beige adipocytes. Adult humans possess mitochondria-enriched beige-like adipocytes that display molecular signatures resembling murine beige fat and can be reactivated by cold or b3 agonists causing metabolic benefits. Thermogenic activity in specialized adipose cells depends on the fitness of mitochondrial organelles carrying uncoupling respiration or futile reactions that dissipate energy as heat. Mitochondrial respiration occurs in organized structures called cristae, tubular invaginations of the inner mitochondrial membrane that function as battery-like devices generating and dissipating energy. We have identified a new cold stress inducible mechanism that controls mitochondrial cristae assembly and thermogenic activity in brown/beige adipose cells. Components of this thermogenic regulatory mechanism include the cold- and adrenergic-activated ER resident kinase PERK that signals to mitochondrial protein import machinery facilitating assembly of MICOS complexes that organize and promote cristae biogenesis. In vitro and in vivo studies show that adipose PERK deficiency results in defective cristae formation and impaired thermogenic responses. The premise of this application is that the ER signals to the mitochondrial protein import to control cristae biogenesis and form competent thermogenic adipocytes protecting against lower temperatures and obesity/T2D. We have three aims: 1) determine the regulatory mechanisms of cristae biogenesis and thermogenic function through PERK activation, focusing on how PERK controls cristae formation including activation of OGT-dependent glycosylation; 2) determine the mechanisms of cold-dependent mitochondrial protein import coupled to thermogenic function, investigating co-chaperones and TOM70-assisted MIC19 protein import that causes cristae biogenesis and thermogenic function, and 3) analysis of mitochondrial cristae formation and metabolic/energetic function during cold- and diet-induced thermogenesis using genetic mouse models, focusing on how different this signaling ER- mitochondria axis impacts energy balance and metabolism during cold adaptation and high fat diet feeding. The outcomes of this application will determine the regulatory mechanisms th...