Persistent mTORC1 activation underlies sex dimorphic progression of MASLD in mice with hepatocyte prohibitin-1 deficiency

Abstract Prohibitins (PHB1,2) are highly conserved lipid-raft associated proteins that physically interact to form a multimeric ring supercomplex in mitochondrial and plasma membranes where they are intimately involved in regulating cellular metabolism. Prior studies in disparate cell models have implicated PHB1 as a mediator of insulin signaling and its downstream effector, the mechanistic target of rapamycin complex 1 (mTORC1), but the mechanisms and physiological implications of these interactions are unclear. Here, we examined the role of PHB1 in regulating insulin and nutrient mediated activation of mTORC1 in liver using genetic and pharmacological approaches in mice and hepatocyte culture. Interestingly, male mice with hepatocyte-specific PHB1 haploinsufficiency (hPHB1-KD) at 6 months displayed features consistent with metabolic dysfunction-associated steatotic liver disease (MASLD), characterized by liver steatosis and impaired glucose tolerance with hyperinsulinemia, while these parameters were unaffected or even mildly improved in age-matched hPHB1-KD females. Both sexes of hPHB1-KD mice displayed increased basal phosphorylation of mTORC1 and its downstream targets (S6, 4EBP1) in liver compared with WT in fasted state, with minimal responsiveness to insulin. Transcriptomic data revealed strong upregulation of Lpin1 gene in male hPHB1-KD mice, a phosphatidic acid phosphatase regulated by mTORC1 that critically regulates hepatic lipid metabolism. Integrated transcript-/metabolomic analysis showed enriched glycerolipid metabolism and upregulation of MASLD pathway in the liver of hPHB1-KD males. Parallel experiments in AML12 hepatocytes confirmed that PHB1 knockdown causes hyper-activation of mTORC1 signaling, increased cytoplasmic lipin-1 expression and localization, and increased lipid droplet formation. Furthermore, one week of treatment with mTORC1 inhibitor Torin1 reduced hepatic triglycerides and normalized mTORC1 signaling in hPHB1-KD males to levels comparable with WT. Collectively, these findings demonstrate that PHB1 is essential for maintaining metabolic homeostasis in liver via control of mTORC1-lipin1 axis, and further confirm that metabolic effects of PHB1 deficiency in liver are sexually dimorphic.