Development and Characterisation of a Starvation‐ and Exercise‐Induced Murine Model of Microvesicular Steatosis Relevant to Anorexia Nervosa

ABSTRACT Anorexia nervosa (AN) leads to severe dietary restriction, low body weight, and in many patients, hepatocellular steatosis. Clinically, hepatic fat in AN is usually macrovesicular, whereas microvesicular steatosis is more often associated with severe starvation and mitochondrial dysfunction. The precise mechanisms underlying starvation‐associated microvesicular steatosis in AN, however, remain unclear. We therefore developed a murine model that combines food restriction with excessive activity to mimic the metabolic stress of AN and compared it with simple starvation, high‐fat diet (HFD), and normal diet controls. Five‐week‐old female C57BL/6 mice were assigned to food restriction (FR, n = 18), AN ( n = 13), HFD ( n = 11), or control (Ctr, n = 11) groups. FR and AN mice underwent time‐restricted feeding until achieving approximately 25% body weight loss; AN mice were additionally housed with activity wheels. Body weight was monitored throughout. On Day 17, serum and liver samples were collected for biochemistry, histology (H&E, Oil Red O, adipophilin immunostaining and electron microscopy) and exploratory RNA‐seq analysis of metabolic pathways, including β‐oxidation and lipid transport. Two FR mice died during the experiment, whereas no deaths occurred in the AN, HFD or Ctr groups. Both FR and AN groups showed marked weight loss compared with Ctr ( p < 0.05). Histology revealed focal small‐droplet steatosis in a subset of AN mice and large‐droplet steatosis in HFD mice, whereas FR mice showed minimal steatosis. Oil Red O staining confirmed small‐droplet fat in 23.1% of AN mice and also demonstrated lipid accumulation in HFD mice. Adipophilin‐positive areas were significantly increased in FR, AN and HFD groups versus Ctr ( p < 0.05), although no significant difference was detected between AN and FR. Biochemically, FR and AN mice exhibited lower TG and Glu and higher AST compared with Ctr ( p < 0.05). RNA‐seq showed clustering of AN and FR, distinct from HFD and Ctr, and suggested alterations in pathways related to fatty‐acid oxidation and lipid handling. We established a starvation‐ and exercise‐associated AN model that reproduces early, modest microvesicular steatosis and metabolic liver injury. Given the short observation period and limited extent of steatosis, RNA‐seq findings should be regarded as preliminary and hypothesis‐generating. Prolonged protocols and direct assessment of β‐oxidation and lipid transport will be required to build a more robust disease model.