Triacylglycerol hydrolysis and fatty acid partitioning in the liver

University of Minnesota M.S. thesis. May 2013. Major: Nutrition. Advisor: Douglas G. Mashek. 1 computer file (PDF); iii, 83 pages.The liver is a vital organ for the homeostatic control of both carbohydrate and lipid metabolism, functioning as a major regulatory site of energy storage, processing, and transformation. This project focused on liver lipid metabolism--it's production and trafficking of very low density density lipoproteins in particular. Specifically, we investigated whether VLDL trafficking is affected by 1) glucose and/or insulin, and 2) source of fatty acid (de novo or exogenous). As these were two distinct questions, each required its own sub-study, both of which are outlined separately below. Although it is well known that glucose and insulin influence the metabolism of fatty acids (FA) and the secretion of very low-density lipoprotein (VLDL), little is understood about how they regulate the partitioning of FA hydrolyzed from hepatic stored triacylglycerol (TAG) to different metabolic pathways. The aims of this study were to investigate the effects of differing concentrations of glucose and insulin on TAG hydrolysis and the subsequent partitioning of FA to pathways of secretion and oxidation in primary hepatocytes. We utilized primary mouse hepatocytes for pulse-chase experiments using [1-14C] oleate to examine the turnover and partitioning of the labeled FA. In addition to looking at the effects of differing glucose and insulin concentrations, we wanted to investigate whether any effects differed following acute (6 h) vs. chronic (24 h) treatment. Although we did not observe any acute effects of either glucose or insulin on TAG hydrolysis or partitioning of hydrolyzed FA, chronic insulin exposure decreased both FA oxidation and TAG secretion. These results suggest that insulin and glucose do not acutely influence TAG hydrolysis or channeling of hydrolyzed FA, but longer exposure to insulin reduces FA oxidation and secretion. There is a great deal of evidence suggesting that before being incorporated into VLDL for secretion, hepatic FA are first directed to storage in the cytosolic lipid droplet pool, and must subsequently be mobilized and channeled to assembly into VLDL. It remains unclear, however, whether the liver stores FA from different sources in distinct lipid droplet pools. Furthermore, should distinct storage pools exist, it is unknown whether they are preferentially channeled to different pathways, such as VLDL assembly. We performed experiments utilizing [1-14C] acetate and [1-14C] palmitate to represent FA derived from de novo lipogenesis and exogenous uptake, respectively. There was no difference between percentage of cellular acetate (via conversion to long chain FA) or palmitate secreted into the media at 4, 8, and 16 h in primary hepatocytes. However, we consistently saw a greater percentage of the label from acetate (de novo FA) secreted into the media after 2 h. Following the same 2 h labeling period in primary hepatocytes, we observed no difference between the partitioning of [1-14C] acetate and [1-14C] palmitate to the microsomal (ER) and lipid droplet fractions. Finally, to examine in vivo partitioning of the different FA, we injected mice with labeled FA and isolated their livers to quantify the contribution of the respective FA to cellular TAG and phospholipids. There was no difference in the incorporation of differentially sourced FA in liver TAG or phospholipid fractions. The results of these studies suggest that given the choice between FA taken up from exogenous sources and FA synthesized de novo, the liver may acutely preferentially secrete FA from de novo synthesis, however more work is needed to confirm this finding