Fatty Acid Cycling in Human Hepatoma Cells and the Effects of Troglitazone

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- 13 Fatty acids extracted from cell pellets after saponification were analyzed by gas chromatography/mass spectrometry. Peroxisomal ␤-oxidation of uniformly 13 C-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 ␤-oxidation of C18 to C16 fatty acids, and the interconversion of long chain fatty acids was associated with reduced de novo lipogenesis.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. The ␤-oxidation system of the peroxisomes, unlike that of the mitochondria, is not coupled to oxidative phosphorylation and is an important source of acetyl (2-carbon) units for the synthesis of long chain fatty acids by chain elongation (1). Fatty acid cycling of polyunsaturated fatty acids in the peroxisomes has been shown to play an important role in the metabolism of essential fatty acids (2). The role of fatty acid cycling by chain shortening/elongation of saturated fatty acids is not well known. 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.Recently, 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). For example, chain shortening of [U-13 C]stearate produces palmitate with a mass shift of 16 daltons due to 13 C carbons, and the elongation of [U-13 C]stearate produces arachidate (C20:0) and behenate (C22:0) with a characteristic mass shift of 18 daltons. Thus, chain shortening and elongation can be measured by the formation of these unique isotopomer species. 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 proliferat...

The Role of the Novel Lipokine Palmitoleic Acid in Health and Disease

“…Because dietary concentrations of palmitoleate are relatively low, and it is rapidly oxidized (15,25), it is possible that palmitoleate plasma concentrations are affected by endogenous production from dietary vaccenic acid. Vaccenic acid is the most predominant ruminant trans FA, and its metabolic effects are either neutral or suggest protection against atherosclerosis and activation of PPARs (23,(26)(27)(28)(29). Thus, because vaccenic acid is a precursor of trans-palmitoleate, the chance of increased metabolic benefits through dairy consumption arises.…”

“…Lipogenesis is mediated by stearoyl-CoA desaturase 1 (SCD1) 6 , the rate-limiting enzyme catalyzing the synthesis of MUFAs, mainly oleate (18:1c9) and palmitoleate (16:1c9) (22). In addition to palmitate, oleate also can be converted into palmitoleate by chain shortening in human hepatocellular carcinoma cells (23). In humans, cis-palmitoleate biosynthesis occurs primarily in the liver, and secondarily in adipose tissue, where it is later incorporated into phospholipids, TGs, waxes, and cholesterol esters.…”

Compartmentalization of stearoyl-coenzyme A desaturase 1 activity in HepG2 cells

“…corresponding to the fraction of molecules that contain 0, 1, 2, 3, ….. 13 C substitutions. The design of the study using [1,[2][3][4][5][6][7][8][9][10][11][12][13] C]acetate and [U- 13 C]stearate or [U- 13 C]palmitate allowed the separation of three separate pools for each fatty acid. The added (exogenous) fatty acid pool was represented by the U-13 C-labeled fatty acid (M118 or M116), the newly synthesized fatty acid pool was represented by the fatty acid with mass shift of M12 and M14, and fatty acids made from the preexisting fatty acid pool were represented by the "unlabeled" (M10) fatty acid ( Fig.…”

“…These calculations used information from the "light" fraction of molecules with peaks corresponding to m 2 , m 4 , and m 6 molecules from incorporation of [1,[2][3][4][5][6][7][8][9][10][11][12][13] C] acetyl-CoA in de novo synthesis of palmitate. The contribution of de novo palmitate synthesis ranged from 51.3% to 53.8% ( …”

“…The effects of SCD1 inhibition on these dynamics in fatty acid metabolism are poorly understood. Stable isotopes such as the uniformly labeled fatty acids [U- 13 C]stearate and [U- 13 C]palmitate have been used to study LCFA desaturation, chain elongation, and shortening (12,13). In this study, stable isotopes were used to distinguish between the SCD1 pathways of palmitate-topalmitoleate and stearate-to-oleate conversion as well as chain elongation and chain shortening.…”