Abstract
Fatty acid cycling by chain shortening/elongation in the peroxisomes is an important source of fatty acids for membrane lipid synthesis. Its role in the homeostasis of nonessential fatty acids is poorly understood. We report here a study on the cycling of saturated fatty acids and the effects of troglitazone in HepG2 cells in culture using [U-13C]stearate or [U-13C]oleate and mass isotopomer analysis. HepG2 cells were grown in the presence of 0.7 mmol/liter [U-13C]stearate or [U-13C]oleate, and in the presence and absence of 50 microM troglitazone for 72 h. Fatty acids extracted from cell pellets after saponification were analyzed by gas chromatography/mass spectrometry. Peroxisomal beta-oxidation of uniformly 13C-labeled stearate (C18:0) and oleate (C18:1) resulted in chain shortening and produced uniformly labeled palmitate (C16:0) and palmitoleate (C16:1). In untreated cells, 16% of C16:0 was derived from C18:0 and 26% of C16:1 from C18:1 by chain shortening. Such contributions were significantly increased by troglitazone to 23.6 and 36.6%, respectively (p < 0.001). Desaturation of stearate contributed 67% of the oleate, while reduction of oleate contributed little to stearate (2%). The desaturation of C18:0 to C18:1 was not affected by troglitazone. Our results demonstrated a high degree of recycling of C18:0 and C18:1 to C16:0 and C16:1 through chain shortening and desaturation. Chain shortening was accompanied by chain elongation in the synthesis of other long chain fatty acids. Troglitazone specifically increased recycling by peroxisomal beta-oxidation of C18 to C16 fatty acids, and the interconversion of long chain fatty acids was associated with reduced de novo lipogenesis.
Highlights
The peroxisomes and the mitochondria are two separate fatty acid -oxidation systems having distinct roles in fatty acids catabolism, energy production, and substrate cycling within the cell
We report here a study of chain shortening and elongation of stearate (C18:0) and the role of activation of peroxisome oxidation with troglitazone, a peroxisome proliferator-activated receptor (PPAR␥)1 ligand, on stearate metabolism in HepG2 cells in culture using uniformly 13C-labeled stearate and oleate and mass isotopomer analysis
We found that the precursor enrichments as determined from arachidate were much higher than those estimated from the palmitate isotopomer ratios (Fig. 5 and Table I) suggesting that chain elongation and de novo lipogenesis may have different precursor pools within the cells
Summary
The peroxisomes and the mitochondria are two separate fatty acid -oxidation systems having distinct roles in fatty acids catabolism, energy production, and substrate cycling within the cell. Because of recycling of label and the lack of proper isotopic methods, the study of chain shortening/elongation of nonessential fatty acids has been difficult. We have developed stable isotope methods for the study of essential and nonessential fatty acid metabolism using uniformly labeled compounds and mass spectrometry [3, 4]. We report here a study of chain shortening and elongation of stearate (C18:0) and the role of activation of peroxisome oxidation with troglitazone, a peroxisome proliferator-activated receptor (PPAR␥) ligand, on stearate metabolism in HepG2 cells in culture using uniformly 13C-labeled stearate and oleate and mass isotopomer analysis
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