Studies on the assembly of cytosolic lipid droplets. Importance of Phospholipase D and ADRP in the process

Abstract

The storage of triglycerides in cells such as adipocytes, liver or muscle cells is of great physiological importance, having implications for our most important metabolic diseases such as obesity, the metabolic syndrome and type II diabetes. A common denominator in these diseases is the insulin resistance, which is also highly related to the production of atherogenic lipoproteins and the development of atherosclerosis and consequently cardio-vascular diseases. Of central importance for the development of insulin resistance is the accumulation of triglycerides as lipid droplets in liver and muscles cell. Such an accumulation is dependent on the protein ADRP (adipocyte differentiation related protein). This protein could therefore have a central role in the development insulin resistance. Relatively little was known about the process by which these ADRP containing lipid droplets are formed. To address this issue, we first developed a cell-free system that reproduces the early stages of lipid droplet formation. We could provide for the first time evidence that the early lipid droplets originate from microsomal membranes. We observed how lipid droplet associated proteins, such as ADRP, caveolin, vimentin and GRP78, were budding with lipid droplets from microsomal membranes. We identified the presence of a strong activator to the assembly of lipid droplets and partially purified this activator. A novel and important observations was made: the enzyme Phospholipase D (PLD) was necessary for the assembly of the lipid droplets. We verified the importance of PLD1 for the formation of lipid droplets in intact cells. The used of siRNA towards PLD1 led to a decrease in the amount of lipid droplets. Conversely over-production of PLD1 gave rise to an increased formation of lipid droplets. Depletion of PLD1 led to a diminished expression of ADRP, while the over-expression of PLD1 caused an increase expression of ADRP in the cells. These and other results indicate that the effect of PLD1 on the assembly of lipid droplets is mediated via an increased cellular level of ADRP. Interestingly, we also demonstrated that the newly formed lipid droplets could grow by a fusion process. Our findings demonstrated that a major constituent of green tea, Epigallocathechine gallate (EGCG), which has the ability to activate PLD, also induces the formation of lipid droplets in liver cells. Furthermore, EGCG seems to divert triglycerides from the assembly of the very low density lipoproteins (VLDL), towards lipid droplets, thereby decreasing VLDL secretion and promoting the intracellular degradation of its structural protein apoB-100. We propose that lipid droplets are assembled in two steps; the budding of primordial droplet under the influence of PLD1 and ADRP and a fusion of these primordial droplets to form the mature droplets.