Identification of Factors Regulating Cytoplasmic and Nuclear Lipid Droplets

Abstract

Lipid droplets (LDs) are cellular metabolic energy reservoirs composed of a neutral lipid core of triglycerides (TG) and cholesterol esters (CE) that is surrounded by a surface monolayer of phospholipids and embedded proteins. The most quantitatively significant phospholipid in eukaryotic cellular membranes is phosphatidylcholine (PC), which is de novo synthesized by the CDP-choline pathway under the control of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT) α. In the liver and intestine, the neutral lipids in LDs are hydrolyzed, re-esterified and assembled into lipoproteins for secretion. Increased PC synthesis is required for LD biogenesis and hepatic lipoprotein secretion. The aim of this project was to investigate mechanisms involved in regulating TG and PC biosynthesis during LD biogenesis and cellular lipid metabolism. Knockout of CCTα in intestinal-derived Caco2 cells (CCTα-KO cells) was used to determine how PC metabolism controls TG storage in LDs and secretion in chylomicrons. CCTα-KO cells had significantly decreased de novo PC synthesis and number of cytosolic LDs (cLDs), but increased droplet size and TG mass. Secretion of chylomicrons was reduced in differentiated CCTα-KO cells, indicating a specific requirement for the CDP-choline pathway in intestinal cell TG storage and secretion in lipoproteins. CCTα-KO cells also had a significant reduction in nuclear LDs (nLDs), which have CCTα and promyelocytic leukemia nuclear bodies (PML-NBs) coating the surface. Using PML knockout U2OS cells, we show that PML-NBs are critical for regulating the size and number of cLDs and nLDs in response to exogenous fatty acids. PML deficiency diminished both cLDs and nLDs, prevented recruitment of CCTα and LIPIN-1 to nLDs, and reduced PC and TG synthesis. Our research highlights the importance of coordinated regulation of PC and TG metabolism for the formation and function of LDs in the cytoplasm and nucleus.