As key directors of intracellular trafficking, phosphotidylinsotides (PIs) are a family of low abundance lipids that impact almost every process within the eukaryotic cell. The low-density lipoprotein receptor (LDLR), a major determinant of blood LDL levels, is the most efficacious therapeutic target for the prevention of cardiovascular disease. Upon LDL binding, LDLR at the cell surface is internalized, where it releases the bound LDL and then recycles back to the cell surface or is directed to the lysosome for degradation. Recently, we found that knock- down of transmembrane protein 55B (TMEM55B), a phosphatidylinositol (4,5) bisphosphate [PI(4,5)P] phosphatase, stimulated decay of LDLR protein in cells and raised plasma LDLC in mice. Since PI(4,5)P is involved in lysosome formation, we hypothesize that PI(4,5)P phosphatases (such as TMEM55B) may alter LDLC by regulating LDLR intracellular trafficking. We also found that reduction of PI(4,5)P phosphatases caused severely impaired secretion of triglycerides into plasma. Notably, this occurred without the accumulation of liver fat, and interestingly, was observed only in male mice, with no effect in females. The lack of hepatic fat accumulation may be attributed to a number of different mechanisms including increased lipophagy (a process of selective autophagy in which lipid droplets are targeted for lysosomal decay). PI(4,5)P is an important regulator of lysosome reformation after autophagosome fusion, and we found that TMEM55B knock-down increased lysosomes. These findings suggest that PI(4,5)P phosphatases may modulate hepatic TG secretion and storage through a lysosome-dependent mechanism. Thus, the overall objective of this proposal is to evaluate the role of PI(4,5)P in LDLR recycling and TG secretion.
In Aim 1 we will i) Test if PI(4,5)P metabolizing enzymes impact plasma LDL in the absence of LDLR using genetically modified mouse models; ii) Evaluate the effect of modifying PI(4,5)P on Ldlr intracellular levels and localization in mouse hepatocytes; iii) Test if expression of enzymatically inactive Tmem55b rescues the phenotypes observed in the Tmem55b knockout mouse; and iv) Evaluate if PI(4,5)P phosphatase regulation of LDLR recycling is dependent on known regulators of LDLR decay.
In Aim 2 we will i) Compare TG secretion in whole body versus liver-specific Tmem55b knockout male mice to confirm that the defect is a hepatic mechanism; ii) Test if lack of hepatic fat accumulation, as would be expected to result from impaired TG secretion, in hepatocytes from Tmem55b knockout mice is due to changes in fatty acid oxidation, synthesis, or uptake, or TG synthesis; iii) Determine PI(4,5)P phosphatases depend on lysosome and/or autophagosomes to impair TG secretion; and iv) Evaluate if the sex-specific effect of PI(4,5)P modulation of TG secretion is due to the sex chromosome complement (XX vs. XY) and/or gonadal hormones. Investigation of the role of PI(4,5)P in lipid and lipoprotein metabolism may lead to the development of new therapeutic options for reducing cardiometabolic disease risk.
The overall goal of this proposal is to use cellular and animal models to investigate a new pathway that regulates levels of circulating cholesterol and of liver triglycerides. Elevated plasma cholesterol and accumulation of liver fat are major risk factors for heart disease and non-alcoholic fatty liver disease, two metabolic disorders that impact a significant number of Americans. !
| Kim, Kyungpil; Theusch, Elizabeth; Kuang, Yu-Lin et al. (2018) ZNF542P is a pseudogene associated with LDL response to simvastatin treatment. Sci Rep 8:12443 |
