Impact of Fabp1/Scp-2/Scp-x gene ablation (TKO) on hepatic phytol metabolism in mice

Abstract

Studies in vitro have suggested that both sterol carrier protein-2/sterol carrier protein-x (Scp-2/Scp-x) and liver fatty acid binding protein [Fabp1 (L-FABP)] gene products facilitate hepatic uptake and metabolism of lipotoxic dietary phytol. However, interpretation of physiological function in mice singly gene ablated in the Scp-2/Scp-x has been complicated by concomitant upregulation of FABP1. The work presented herein provides several novel insights: i) An 8-anilino-1-naphthalenesulfonic acid displacement assay showed that neither SCP-2 nor L-FABP bound phytol, but both had high affinity for its metabolite, phytanic acid; ii) GC-MS studies with phytol-fed WT and Fabp1/Scp-2/SCP-x gene ablated [triple KO (TKO)] mice showed that TKO exacerbated hepatic accumulation of phytol metabolites in vivo in females and less so in males. Concomitantly, dietary phytol increased hepatic levels of total long-chain fatty acids (LCFAs) in both male and female WT and TKO mice. Moreover, in both WT and TKO female mice, dietary phytol increased hepatic ratios of saturated/unsaturated and polyunsaturated/monounsaturated LCFAs, while decreasing the peroxidizability index. However, in male mice, dietary phytol selectively increased the saturated/unsaturated ratio only in TKO mice, while decreasing the peroxidizability index in both WT and TKO mice. These findings suggested that: 1) SCP-2 and FABP1 both facilitated phytol metabolism after its conversion to phytanic acid; and 2) SCP-2/SCP-x had a greater impact on hepatic phytol metabolism than FABP1.

Keywords: fatty acid binding protein 1/sterol carrier protein-2/sterol carrier protein-x; peroxisomal oxidation; triple knockout.

Fig. 1.

Displacement of FABP1 and SCP-2 bound ANS by phytol versus phytanic acid. FABP1 (500 nM, closed circle) with ANS (35 μM) or SCP-2 (500 nM, open circle) with ANS (50 μM) were titrated with increasing amounts of phytol (A) or phytanic acid (B). ANS fluorescence remaining was measured by scanning 410–600 nm with excitation at 380 nm as described in the Materials and Methods. Values represent average percent remaining ANS fluorescence ± standard error (n = 4–5).

Fig. 2.

Hepatic accumulation of branched-chain, nonbranched, and total fatty acids in WT and TKO mice fed dietary phytol. Female (A, C, E) and male (B, D, F) WT (open bars) and Fabp1/Scp-2/Scp-x-null (TKO) (black bars) mice were fed control or 0.5% phytol-supplemented chow as described in the Materials and Methods. Hepatic levels of branched-chain FA (A, B), FA (non-branched) (C, D), and total FA (E, F) were determined as described in the Materials and Methods. Values represent the mean nanomoles if fatty acid per milligrams total liver protein ± SE (n = 4–6); ND, not detected; *P ≤ 0.05 versus WT on control-diet; $P ≤ 0.05 versus TKO on control-diet; @P ≤ 0.05 versus WT on phytol-diet.

Fig. 3.

Mass distribution of phytol metabolites in hepatic lipids of WT and TKO mice fed dietary phytol. Female (A, C, E) and male (B, D, F) WT (white bars) and Fabp1/Scp-2/Scp-x-null (TKO) (black bars) mice were fed control or 0.5% phytol-supplemented chow as described in the Materials and Methods. Liver lipids were extracted and analyzed by GC-MS to determine the contents (nanomoles per milligram protein) of phytanic acid (A, B), pristanic acid (C, D), and Δ2,3-pristenic acid (E, F) as described in the Materials and Methods. Values represent the mean nanomoles of fatty acie per milligram of total liver protein ± SE (n = 4–8); ND, not detected; *P ≤ 0.05 versus WT control-diet; $P ≤ 0.05 versus TKO control-diet; @P ≤ 0.05 versus WT phytol-diet.

Fig. 4.

Relative proportion of phytol metabolites as percent of total branched-chain fatty acid content. Liver lipids were extracted and analyzed by GC-MS to determine the relative proportions of phytanic acid (A, B), pristanic acid (C, D), and Δ2,3-pristenic acid (E, F) as described in the Material and Methods. Values represent the mean percent of total liver branched chain fatty acid ± SE (n = 4–6); ND, not detected; *P ≤ 0.05 versus WT control-diet; $P ≤ 0.05 versus TKO control-diet; @P ≤ 0.05 versus WT phytol-diet.

Fig. 5.

Mass distribution of fatty acid subgroups in livers of WT and TKO mice fed dietary phytol. Female (A, C, E, G) and male (B, D, F, H) WT (white bars) and Fabp1/Scp-2/Scp-x-null (TKO) (black bars) mice were fed control or 0.5% phytol-supplemented chow as described. Liver lipids were extracted and quantities (nanomoles per milligram of protein) of total hepatic saturated (Sat) fatty acids (A, B), unsaturated (Unsat) fatty acids (C, D), MUFAs (E, F), and PUFAs (G, H) determined by GC-MS as described in the Materials and Methods. Values represent the mean nanomoles of fatty acid per milligram of total liver protein ± SE (n = 4–6); *P ≤ 0.05 versus WT control-diet; $P ≤ 0.05 versus TKO control-diet; @P ≤ 0.05 versus WT phytol-diet.

Fig. 6.

Effect of TKO on hepatic fatty acid subgroup ratios and peroxidizability indices in phytol-fed mice. Female (A, C, E) and male (B, D, F) WT (white bars) and Fabp1/Scp-2/Scp-x-null (TKO) (black bars) mice were fed control or 0.5% phytol-supplemented chow. Ratios of saturated fatty acid to unsaturated fatty acid (Sat/Unsat) (A, B) and PUFAs/MUFAs (C, D) were calculated using the corresponding hepatic fatty acid subgroup values in this figure. Peroxidizability indices (E, F) were calculated as described in the Materials and Methods and presented in the same format. Values represent the mean ratio (or index) ± SE (n = 4–6); *P ≤ 0.05 versus WT control-diet; $P ≤ 0.05 versus TKO control-diet; @P ≤ 0.05 versus WT phytol-diet.

Fig. 7.

Schematic of proposed FABP1 and SCP-2/SCP-x interactions with the phytol metabolic pathway. FABP1 and SCP-2, both present at high level in hepatic cytoplasm, bind phytol metabolites, but not phytol. In this hypothetical scheme, FABP1 and SCP-2 bind phytanic acid and phytenic acid (formed from phytol in the endoplasmic reticulum) and transport them to the peroxisome. At the peroxisomal membrane the phytanic and phytenic acid are converted to their respective CoA thioesters, internalized and desorbed into the peroxisomal matrix wherein they are bound (20, 21) by peroxisomal localized SCP-2 (–11) and potentially FABP1 (34). Within the peroxisomal matrix, SCP-2 directly interacts with oxidative enzymes and stimulates phytanoyl-CoA 2-hydroxylase, the essential enzyme mediating the first step in peroxisomal α-oxidation of branched-chain fatty acids (8, 19). SCP-x, an exclusively peroxisomal protein, is the only known branched-chain 3-ketoacyl CoA thiolase (, –24). Successive cycles of α- and β-oxidation shortens the branched-chain fatty acids to yield short-chain (