Distinct yet Overlapping Functions of VMP1 and TMEM41B in Modulating Hepatic Lipoprotein Secretion and Autophagy in MASH.

BACKGROUND

Transmembrane protein 41B (TMEM41B) and vacuolar membrane protein 1 (VMP1) are endoplasmic reticulum (ER) transmembrane scramblase proteins that have been recently identified to have important roles in autophagy and hepatic lipoprotein secretion. While TMEM41B and VMP1 are structurally and functionally similar, the nature of their interactions and how they coordinately regulate hepatic lipoprotein secretion and autophagy in metabolic-associated steatotic liver disease (MASLD) and metabolic-associated steatohepatitis (MASH) remains unclear.

METHODS

Liver-specific and hepatocyte-specific knockout (KO) mice as well as knock-in (KI) mice were generated from or mice by crossing with albumin-Cre mice or by injecting AAV8-TBG-cre, respectively. Lipid metabolism in these mice was characterized by lipidomic analyses. Mice with hepatic overexpression of TMEM41B that were fed a MASH diet were also characterized. To explore the relationship between TMEM41B and VMP1, double KO (DKO), KO/ KI, and KO/ KI mice were generated, and steatosis and autophagy were characterized.

RESULTS

The loss of hepatic severely impaired very low-density lipoprotein (VLDL) secretion, resulting in significant microsteatosis, increased hepatic triglycerides, inflammation, fibrosis, and ultimately the MASH development. TMEM41B protein was decreased in human MASLD livers. Overexpression of TMEM41B mitigated the effects of diet-induced MASLD. Mice lacking both and (DKO) showed further impairment in VLDL secretion compared to single KO, but were similar that of KO mice. Lipidomic analysis of liver tissues revealed decreased levels of phosphatidylcholine and phosphatidylethanolamine, along with increased neutral lipids. Cellular fractionation studies indicated that VMP1 and TMEM41B localize at the mitochondrial-associated membrane (MAM). Electron microscopy analysis showed reduced contact between mitochondria and the ER in hepatocytes deficient in either VMP1 or TMEM41B. The loss of hepatic VMP1 or TMEM41B led to markedly increased levels of LC3B-II and p62/SQSTM1, which were not further affected by double deletion of VMP1 and TMEM41B. Restoring VMP1 in KO mice partially improved defective VLDL secretion, though autophagy was only partially corrected by overexpression of VMP1 at a low but not high level. In contrast, restoring TMEM41B in KO mice dose-dependently improved both defective VLDL secretion and autophagy.

CONCLUSION

Loss of hepatic VMP1 or TMEM41B decreases MAM and phospholipid content and reduces VLDL secretion, resulting in the development of MASH. TMEM41B and VMP1 may have overlapping but distinct mechanisms in regulating lipoprotein secretion and autophagy.