Compartmentalization of stearoyl-coenzyme A desaturase 1 activity in HepG2 cells - PubMed (original) (raw)

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

Jennifer K Yee et al. J Lipid Res. 2008 Oct.

Abstract

Stearoyl-coenzyme A desaturase 1 (SCD1) catalyzes the conversion of stearate (18:0) to oleate (18:1n-9) and of palmitate (16:0) to palmitoleate (16:1), which are key steps in triglyceride synthesis in the fatty acid metabolic network. This study investigated the role of SCD1 in fatty acid metabolism in HepG2 cells using SCD1 inhibitors and stable isotope tracers. HepG2 cells were cultured with [U-(13)C]stearate, [U-(13)C]palmitate, or [1,2-(13)C]acetate and (1) DMSO, (2) compound CGX0168 or CGX0290, or (3) trans-10,cis-12 conjugated linoleic acid (CLA). (13)C incorporation into fatty acids was determined by GC-MS and desaturation indices calculated from the respective ion chromatograms. FAS, SCD1, peroxisome proliferator-activated receptor alpha, and peroxisome proliferator-activated receptor gamma mRNA levels were assessed by semiquantitative RT-PCR. The addition of CGX0168 and CGX0290 decreased the stearate and palmitate desaturation indices in HepG2 cells. CLA led to a decrease in the desaturation of stearate only, but not palmitate. Comparison of desaturation indices based on isotope enrichment ratios differed, depending on the origin of saturated fatty acid. SCD1 gene expression was not affected in any group. In conclusion, the differential effects of SCD1 inhibitors and CLA on SCD1 activity combined with the dependence of desaturation indices on the source of saturated fatty acid strongly support the compartmentalization of desaturation systems. The effects of SCD1 inhibition on fatty acid composition in HepG2 cells occurred through changes in the dynamics of the fatty acid metabolic network and not through transcriptional regulatory mechanisms.

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Figures

Fig. 1.

Pathways of SCD1 desaturation. Oleate is made from the desaturation of stearate. Palmitoleate is made from the desaturation of palmitate. Vaccenate is only made by chain elongation of palmitoleate and cannot be made directly from stearate. A: Addition of labeled stearate and labeled acetate allows distinction between the pathways by GC-MS analysis and provides information on chain shortening. B: Alternatively, addition of labeled acetate and labeled palmitate provides information on de novo lipogenesis and chain elongation.

Fig. 2.

Ion chromatogram of fatty acids, with retention times on the x axis and relative abundance on the y axis. Retention times for palmitate, palmitoleate, stearate, oleate, and vaccenate were 6.17, 6.72, 8.8, 9.45, and 9.58 min, respectively.

Fig. 3.

Desaturation indices of oleate-stearate (18:1 n-9/18:0) and palmitoleate-palmitate (16:1/16:0) in [U-13C]stearate (A, C) and [U-13C]palmitate (B, D) experiments. Each bar represents the average and SEM of triplicate cell culture experiments. * P ⩽ 0.001, ** P ⩽ 0.003. In the experiment with [U-13C]stearate, the average oleate-stearate ratio was significantly decreased (P < 0.001). In the experiment with [U-13C]palmitate, the average oleate-stearate ratio was mildly decreased, but not significantly in the CGX0168 (P = 0.14) and CGX290 groups (P = 0.25); it approached significance in the CLA group (P = 0.08). Both experiments showed a significant decrease in the palmitoleate/palmitate desaturation indices with the inhibitors. With [U-13C]stearate, the average palmitoleate-palmitate ratio was significantly decreased in the CGX0168 and CGX0290 groups (P < 0.001), but it was increased in the CLA group (P = 0.0029). With [U-13C]palmitate, the average ratio was significantly decreased for both the CGX0168 group (P = 0.003) and the CGX0290 group (P < 0.001). However, in the CLA group, the difference was not significant, with a trend toward an increased ratio (P = 0.13).

Fig. 4.

Desaturation index for vaccenate-stearate (18:1 n-7/18:0) and the ratio of stearate to palmitate (18:0/16:0) in [U-13C]stearate (A, C) and [U-13C]palmitate (B, D) experiments. Each bar represents the average and SEM of the ratio for triplicate cell culture experiments. * P < 0.001, ** P < 0.01, *** P = 0.053. The average ratio of vaccenate to stearate with [U-13C]stearate was significantly decreased with CGX0168 (P < 0.001), CGX290 (P < 0.001), and CLA (P < 0.001). In the experiment using [U-13C]palmitate, there was also a significant decrease with CGX0168 (P = 0.005) and GCX0290 (P = 0.009). With CLA, the decrease approached significance with P = 0.053. Both experiments showed similar stearate-palmitate ratios in each of the four groups.

Fig. 5.

Mass spectra of stearate and oleate showing uptake of M+18 stearate (A) and conversion to M+18 oleate (B). Uptake of M+16 palmitate and its conversion showed similar M+16 palmitate and M+16 palmitoleate (data not shown). M+0 stearate or palmitate represents mostly preexisting unlabeled fatty acid and a minor fraction of M+0 from the lack of recombination with M+2 acetate in new synthesis.

Fig. 6.

Correlation between the unlabeled oleate/stearate desaturation index and the (M+18 oleate)/(M+18 stearate) ratio (A) and between the unlabeled palmitoleate/palmitate desaturation index and the (M+16 palmitoleate)/(M+16 palmitate) ratio (B). The oleate pool in HepG2 cells is likely relatively large compared with the stearate pool (A). In this particular instance, the (M+18 oleate)/(M+18 stearate) ratio is linearly correlated with the oleate/stearate desaturation index.

Fig. 7.

Gene expression analysis of SCD1, FAS, peroxisome proliferator-activated receptor α (PPARα), and PPARγ by RT-PCR. 18S served as the internal loading control. There was no effect on gene expression from the addition of inhibitors CGX0168, CGX0290, or _trans_-10,_cis_-12 CLA.

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Compartmentalization of stearoyl-coenzyme A desaturase 1 activity in HepG2 cells - PubMed (original) (raw)