Ablating both Fabp1 and Scp2/Scpx (TKO) induces hepatic phospholipid and cholesterol accumulation in high fat-fed mice.

Although singly ablating Fabp1 or Scp2/Scpx genes may exacerbate the impact of high fat diet (HFD) on whole body phenotype and non-alcoholic fatty liver disease (NAFLD), concomitant upregulation of the non-ablated gene, preference for ad libitum fed HFD, and sex differences complicate interpretation. Therefore, these issues were addressed in male and female mice ablated in both genes (Fabp1/Scp2/Scpx null or TKO) and pair-fed HFD. Wild-type (WT) males gained more body weight as fat tissue mass (FTM) and exhibited higher hepatic lipid accumulation than WT females. The greater hepatic lipid accumulation in WT males was associated with higher hepatic expression of enzymes in glyceride synthesis, higher hepatic bile acids, and upregulation of transporters involved in hepatic reuptake of serum bile acids. While TKO had little effect on whole body phenotype and hepatic bile acid accumulation in either sex, TKO increased hepatic accumulation of lipids in both, specifically phospholipid and cholesteryl esters in males and females and free cholesterol in females. TKO-induced increases in glycerides were attributed not only to complete loss of FABP1, SCP2 and SCPx, but also in part to sex-dependent upregulation of hepatic lipogenic enzymes. These data with WT and TKO mice pair-fed HFD indicate that: i) Sex significantly impacted the ability of HFD to increase body weight, induce hepatic lipid accumulation and increase hepatic bile acids; and ii) TKO exacerbated the HFD ability to induce hepatic lipid accumulation, regardless of sex, but did not significantly alter whole body phenotype in either sex.

Sex-dependent impact of Scp-2/Scp-x gene ablation on hepatic phytol metabolism.

While prior studies focusing on male mice suggest a role for sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x; DKO) on hepatic phytol metabolism, its role in females is unresolved. This issue was addressed using female and male wild-type (WT) and DKO mice fed a phytoestrogen-free diet without or with 0.5% phytol. GC/MS showed that hepatic: i) phytol was absent and its branched-chain fatty acid (BCFA) metabolites were barely detectable in WT control-fed mice; ii) accumulation of phytol as well as its peroxisomal metabolite BCFAs (phytanic acid ¼ pristanic and 2,3-pristenic acids) was increased by dietary phytol in WT females, but only slightly in WT males; iii) accumulation of phytol and BCFA was further increased by DKO in phytol-fed females, but much more markedly in males. Livers of phytol-fed WT female mice as well as phytol-fed DKO female and male mice also accumulated increased proportion of saturated straight-chain fatty acids (LCFA) at the expense of unsaturated LCFA. Liver phytol accumulation was not due to increased SCP-2 binding/transport of phytol since SCP-2 bound phytanic acid, but not its precursor phytol. Thus, the loss of Scp-2/Scp-x contributed to a sex-dependent hepatic accumulation of dietary phytol and BCFA.

Loss of L-FABP, SCP-2/SCP-x, or both induces hepatic lipid accumulation in female mice.

Although roles for both sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) and liver fatty acid binding protein (L-FABP) have been proposed in hepatic lipid accumulation, individually ablating these genes has been complicated by concomitant alterations in the other gene product(s). For example, ablating SCP2/SCP-x induces upregulation of L-FABP in female mice. Therefore, the impact of ablating SCP-2/SCP-x (DKO) or L-FABP (LKO) individually or both together (TKO) was examined in female mice. Loss of SCP-2/SCP-x (DKO, TKO) more so than loss of L-FABP alone (LKO) increased hepatic total lipid and total cholesterol content, especially cholesteryl ester. Hepatic accumulation of nonesterified long chain fatty acids (LCFA) and phospholipids occurred only in DKO and TKO mice. Loss of SCP-2/SCP-x (DKO, TKO) increased serum total lipid primarily by increasing triglycerides. Altered hepatic level of proteins involved in cholesterol uptake, efflux, and/or secretion was observed, but did not compensate for the loss of L-FABP, SCP-2/SCP-x or both. However, synergistic responses were not seen with the combinatorial knock out animals-suggesting that inhibiting SCP-2/SCP-x is more correlative with hepatic dysfunction than L-FABP. The DKO- and TKO-induced hepatic accumulation of cholesterol and long chain fatty acids shared significant phenotypic similarities with non-alcoholic fatty liver disease (NAFLD).

Relative contributions of L-FABP, SCP-2/SCP-x, or both to hepatic biliary phenotype of female mice.

Both sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) and liver fatty acid binding protein (L-FABP) have been proposed to function in hepatobiliary bile acid metabolism/accumulation. To begin to address this issue, the impact of ablating L-FABP (LKO) or SCP-2/SCP-x (DKO) individually or both together (TKO) was examined in female mice. Biliary bile acid levels were decreased in LKO, DKO, and TKO mice; however, hepatic bile acid concentration was decreased in LKO mice only. In contrast, biliary phospholipid level was decreased only in TKO mice, while biliary cholesterol levels were unaltered regardless of phenotype. The loss of either or both genes increased hepatic expression of the major bile acid synthetic enzymes (CYP7A1 and/or CYP27A1). Loss of L-FABP and/or SCP-2/SCP-x genes significantly altered the molecular composition of biliary bile acids, but not the proportion of conjugated/unconjugated bile acids or overall bile acid hydrophobicity index. These data suggested that L-FABP was more important in hepatic retention of bile acids, while SCP-2/SCP-x more broadly affected biliary bile acid and phospholipid levels.

Sterol Carrier Protein-2/Sterol Carrier Protein-x/Fatty Acid Binding Protein-1 Ablation Impacts Response of Brain Endocannabinoid to High-Fat Diet.

Brain endocannabinoids (EC) such as arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) primarily originate from serum arachidonic acid (ARA), whose level is regulated in part by a cytosolic ARA-binding protein, that is, liver fatty acid binding protein-1 (FABP1), not expressed in the brain. Ablation of the Fabp1 gene (LKO) increases brain AEA and 2-AG by decreasing hepatic uptake of ARA to increase serum ARA, thereby increasing ARA availability for uptake by the brain. The brain also expresses sterol carrier protein-2 (SCP-2), which is also a cytosolic ARA-binding protein. To further resolve the role of SCP-2 independent of FABP1, mice ablated in the Scp-2/Scp-x gene (DKO) were crossed with mice ablated in the Fabp1 gene (LKO) mice to generate triple knock out (TKO) mice. TKO impaired the ability of LKO to increase brain AEA and 2-AG. While a high-fat diet (HFD) alone increased brain AEA, TKO impaired this effect. Overall, these TKO-induced blocks were not attributable to altered expression of brain proteins in ARA uptake, AEA/2-AG synthesis, or AEA/2-AG degrading enzymes. Instead, TKO reduced serum levels of free ARA and/or total ARA and thereby decreased ARA availability for uptake to the brain and downstream synthesis of AEA and 2-AG therein. In summary, Scp-2/Scp-x gene ablation in Fabp1 null (LKO) mice antagonized the impact of LKO and HFD on brain ARA and, subsequently, EC levels. Thus, both FABP1 and SCP-2 participate in regulating the EC system in the brain.