Role of triacylglycerol hydrolase in hepatic lipid droplet metabolism

The majority of triacylglycerol (TG) utilized for very-low density lipoproteins (VLDL) assembly is derived from lipolysis-reesterification of TG stored in lipid droplets (LDs). At least two types of LDs exist in hepatocytes -- cytosolic LDs (CLDs) and the microsomal-associated luminal LDs (LLDs). An endoplasmic reticulum luminal lipase, triacylglycerol hydrolase (TGH), was shown to participate in mobilizing intracellular TG stored in LDs. However, it is not clear which pool of TG is hydrolyzed by TGH. LLDs are present in the same subcellular compartment as TGH and thus may serve as the substrate pool for this enzyme.;The mechanism by which TGH may regulate the metabolism of CLDs was investigated in wild type and TGH-deficient mouse hepatocytes. It was found that TGH deficiency led to morphological changes in CLDs and diminished the rate at which preformed CLDs obtain newly formed TG (CLD growth). An alternative mechanism for CLD growth was also explored by tracing the incorporation of fluorescent fatty acids analogues into CLDs by live-cell imaging. The results suggested that newly synthesized TG are assembled into LDs through TGH-dependent and TGH-independent mechanisms.;The role of apoE as cofactor for TGH mediated lipolysis was investigated. Obtained results suggested that the absence of apoE in hepatocytes compromised the lipolysis/reesterification process mediated by TGH. Future studies in this direction should further explore the interaction of apoE with TGH and whether the interaction affects the stability of TGH at the lipid-water interface.;Results presented in this thesis describe the isolation and characterization of LLDs from mouse liver microsomes, providing the first biochemical evidence for the presence of these LDs. LLDs differ from CLDs or VLDL particles in both protein and lipid compositions. TGH was found to associate with LLDs, suggesting it may hydrolyze this pool of TG. Other VLDL secretion related proteins were also found on LLDs, including apolipoprotein E and microsomal triglyceride transfer protein. It was determined that LLDs constitute a minor pool of intracellular TG. Thus, quantitatively TG stored in CLDs may provide the major TG source for VLDL assembly. Overexpression of TGH resulted in increased mobilization of not only LLDs but also CLDs.