Abstract
BACKGROUND: Transmembrane protein 41B (TMEM41B) and vacuolar membrane protein 1 (VMP1) are endoplasmic reticulum (ER) scramblases that shuttle phospholipids between the inner and outer leaflets of the ER membrane. Both TMEM41B and VMP1 also play critical roles in regulating hepatic lipoprotein secretion and autophagy. Despite these similarities, whether TMEM41B and VMP1 exhibit different roles in very low-density lipoprotein (VLDL) secretion and autophagy in the pathogenesis of metabolic-associated steatotic liver disease (MASLD) remains unclear. METHODS: We created liver- and hepatocyte-specific single knockout (KO) and double knockout (DKO) mice for Tmem41b and Vmp1 , as well as overexpression knock-in (KI) mice with hepatic overexpression of TMEM41B, Tmem41b KO/ Vmp1 KI, and Vmp1 KO/ Tmem41b KI. We conducted lipidomic, metabolomic, biochemical, and functional studies in these mice, fed either a chow diet or a MASLD diet. RESULTS: TMEM41B protein levels were decreased in the livers of human subjects with MASLD. The loss of hepatic Tmem41b impaired VLDL secretion, resulting in steatosis, inflammation, and fibrosis. Vmp1 KO mice exhibited similar phenotypes to DKO mice, displaying a more severe defect in VLDL secretion and greater liver injury than Tmem41b KO mice. Lipidomic analysis revealed decreased levels of phosphatidylcholine and phosphatidylethanolamine, along with increased neutral lipids in both Tmem41b KO and Vmp1 KO mice; however, these changes were generally more pronounced in Vmp1 KO mice. VMP1 and TMEM41B localized at the mitochondrial-associated membrane (MAM), and a reduction in mitochondria-ER contact was observed in hepatocytes deficient in either VMP1 or TMEM41B. Ultrastructural electron microscopy analysis showed increased accumulation of "lipid droplet" in the ER membrane bilayer and ER lumen in both Vmp1 KO and Tmem41b KO hepatocytes, with greater ER luminal "lipid droplet" accumulation in Tmem41b KO hepatocytes. The loss of hepatic Vmp1 or Tmem41b led to elevated levels of LC3-II and p62, with significantly higher levels of both markers in Vmp1 KO and DKO mouse livers compared to Tmem41b KO mouse livers. Restoring Vmp1 in Tmem41b KO mice partially improved defective VLDL secretion; however, high expression levels of VMP1 did not correct the hepatic autophagy defect. In contrast, restoring Tmem41b in Vmp1 KO mice dose-dependently enhanced both defective VLDL secretion and autophagy. Importantly, overexpression of hepatic TMEM41B mitigated diet-induced MASLD in mice. CONCLUSION: The loss of hepatic Vmp1 or Tmem41b decreases hepatic MAM and phospholipid content, leading to decreased VLDL secretion and promoting MASLD. While VMP1 and TMEM41B have overlapping functions, VMP1 appears to play a more critical role in regulating VLDL secretion and autophagy in mouse livers than TMEM41B.