Cyclophilin D deficiency exacerbates diet-induced obesity and selectively enhances skeletal muscle mitochondrial respiration in male mice.

Mitochondrial dysfunction is a hallmark of obesity-associated metabolic disease, with the mitochondrial permeability transition pore serving as a key regulator of cellular energy homeostasis. Cyclophilin D, encoded by Ppif, is the principal regulatory component of this pore and has been implicated in insulin secretion, adipocyte survival, and whole-body energy balance. However, whether Ppif loss produces a consistent metabolic phenotype when challenged with high fat diet (HFD) exposure, and whether systemic metabolic alterations reflect tissue-specific changes in mitochondrial oxidative capacity, remains unresolved. Here, we characterized male, 21-weeks-old mice with germline Ppif deletion longitudinally over 34 weeks under standard (SD) and HFD using body composition analysis, indirect calorimetry, glucose and insulin tolerance testing and tissue-resolved, Seahorse-based mitochondrial respirometry. Under SD, knockout mice displayed elevated fat mass without differences in food intake or energy expenditure. High-fat feeding progressively amplified this adiposity, with knockout mice developing significantly greater body weight and fat mass than wild-type littermates by weeks 28 and 36. Glucose tolerance and insulin sensitivity remained comparable between genotypes under either diet. ANCOVA revealed significant effects of lean mass (p < 0.001) and genotype (p = 0.017) on energy expenditure, as well as a significant lean mass × genotype interaction (p = 0.025). Consistent with a distinct contribution of lean mass to EE in KO vs. WT mice, we found increased ADP-stimulated mitochondrial respiration in quadriceps of knockout mice under both diets, without corresponding differences in brown adipose tissue or liver, or changes in mitochondrial content across tissues. These findings establish cyclophilin D as a diet- and tissue-specific regulator of energy homeostasis, informing therapeutic strategies targeting the permeability transition pore or cyclophilin D interactome in metabolic disease.