| The liver is a central organ to whole-body metabolism and mediates many of the adaptive responses to changes in nutrient availability, such that the appropriate energy sources are used and blood glucose levels maintained, whether directly after a meal or after a twelve-hour fast. The adaptive responses to fasting in liver are largely mediated by the nuclear receptor peroxisome proliferator-activated receptor alpha, or PPARalpha.;PPARalpha can be activated by a de novo synthesized lipid ligand---6:0/18:1-glycerophosphocholine (16:0/18:1-GPC)---the synthesis of which is dependent on fatty acid synthase (FAS), but little is known about the regulation of this pathway. My thesis focused on post-translational mechanisms controlling endogenous activation of PPARalpha in the liver and used mouse liver and a hepatocyte cell line as model systems.;In addition to its role in PPARalpha activation during fasting, FAS helps store excess calories as fat during feeding. We demonstrated that this paradoxical relationship involves the differential regulation of FAS in at least two distinct subcellular pools: cytoplasmic and membrane-associated FAS, the latter being attached to membranes by a strong peripheral membrane association. To find candidate proteins mediating FAS membrane localization we used a proteomics approach to identify compartment-specific FAS-associated proteins. We identified three proteins---Septin-2, Septin-7, and 40S ribosomal protein S18---that in two different liver model systems associate with FAS exclusively in the membrane fraction. Because the septins are involved in membrane structuring and scaffolding, these proteins may be involved in FAS membrane localization.;The ratio of cytoplasmic to membrane FAS specific activity was increased with fasting or in the absence of insulin, indicating higher cytoplasmic FAS activity under conditions associated with PPARalpha activation. This effect was due to a nutrient-dependent and compartment-selective covalent modification of FAS: cytoplasmic FAS was preferentially phosphorylated during feeding or insulin treatment at Thr-1029 and Thr-1033, which flank a dehydratase domain catalytic residue. Mutating these sites to alanines promoted PPARalpha target gene expression. mTORC1, a mediator of the feeding/insulin signal to induce lipogenesis, emerged as a mediator of FAS phosphorylation, inhibiting cytoplasmic FAS activity and reducing PPARalpha target gene expression in a FAS-dependent manner.;Next, we investigated the role of ligand transport in FAS-mediated PPARalpha activation. 16:0/18:1-GPC is synthesized in the cytoplasm and it is not known how it reaches the nuclear PPARalpha. We identified phosphatidylcholine transfer protein (PCTP) as a possible transport protein for this ligand. PCTP knockdown in Hepa1-6 hepatocytes caused dramatic reductions in expression of PPARalpha target genes, and PCTP co-immunoprecipitated with PPARalpha. Immunofluorescent imaging showed that starvation of cells caused an accumulation of PCTP in the nucleus, consistent with a shuttling function controlled by nutrition. Using mass spectrometry, we demonstrated that PCTP binds 16:0/18:1-GPC. We further showed that the binding of this ligand to PCTP is FAS-dependent: in mice with liver-specific knockout of FAS, the amount of 16:0/18:1-GPC bound to PCTP in the nucleus was significantly reduced.;Together, these findings suggest that multiple modes of post-translational regulation of FAS combined with regulation of lipid delivery by PCTP control fasting-induced PPARalpha activation in liver. |
