Formation of crystalline tripalmitin-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 37 degrees C 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 37 degrees C revealed a multiplicity of brilliant white inclusions between crossed polars in cells incubated with palmitic acid. These birefringent structures exhibited 90 degrees 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 [1-14C]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, respectively, 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.