in-rich spicules in isolated hepatocytes

Conditions were developed for rapid deposition of triglyceride in isolated rat hepatocytes. Liver cells from fasted rats were incubated for 90 min at 37OC with 3.0 mM palmitic or oleic acid, 4 % bovine serum albumin, 20 mM glucose, 10 mM lactate, and 1 mM pyruvate. When oleic acid was used, numerous cytoplasmic lipid droplets were produced. When hepatocytes were incubated with palmitic acid, similar amounts of triglyceride were synthesized but instead of lipid droplets, a vast accumulation of peculiar spicules permeated the cytoplasm. These inclusions appeared in myriads of swirled threads, thick elongated angular plates, and needles, some of which exhibited longitudinal osmiophilic bands of 250 A thickness. These structures were associated with smooth endoplasmic reticulum. The cells appeared otherwise normal. Polarized light microscopy at 37OC revealed a multiplicity of brilliant white inclusions between crossed polars in cells incubated with palmitic acid. These birefringent structures exhibited 90° periodicity between both maximum brilliance and extinction, indicative of anisotropic crystalline deposits. Molecular species analysis of triglycerides in cells incubated with palmitic acid, together with data on [l-'*C]palmitic acid incorporation, demonstrated an almost exclusive synthesis of tripalmitin. Spicules isolated from homogenized hepatocytes displayed needles containing longitudinal single and double osmiophilic bands of 110 A and 260 A thickness, repectively, and lipid spicular aggregates. The isolated spicules were almost pure tripalmitin by analysis. These observations document the formation and development of crystalline triglyceride in living cells and may provide a unique system for the study of cellular lipid synthesis, transport and deposition. -Ontko, J. A., D. L. Stiers, and W. F. Woodside. Formation of crystalline tripalmitin-rich spicules in isolated hepatocytes. J. Lipid Res. 1989. 30: 1375-1384. Supplementary key words birefringence endoplasmic reticulum fatty acids lipid droplets liver oleic acid palmitic acid triglyceride syntheddeposition Triglycerides and cholesteryl esters are extremely hydrophobic lipids that deposit in a wide variety of cellular systems in the form of cytoplasmic lipid droplets (1-7). The relative abundance of triglycerides and cholesteryl esters in these droplets is a function of the cell type and the species of lipid, lipoproteins, and other substrates in the extracellular fluid environment. Lipid droplet subpopulations of different solvent density have been isolated from rat liver (8). In this organ triglycerides and cholesteryl esters are also present in nascent very low density lipoprotein particles in the secretory pathway (7, 9-12). In the course of studies on the metabolism of triglyceride-rich lipid droplets in isolated rat hepatocytes, we noticed a plethora of peculiar crystalline-like structures dispersed throughout the cytoplasm when the cells were incubated with palmitic acid under conditions of rapid triglyceride synthesis. This synthetic process occurs in the outer leaflet of the endoplasmic reticulum bilayer (13). The present communication describes the formation, ultrastructure, and chemistry of these unusual lipid deposits. MATERIALS AND METHODS