Disruption of tumor suppressor gene Hint1 leads to remodeling of the lipid metabolic phenotype of mouse liver

Abstract

A lipidomic and metabolomic investigation of serum and liver from mice was performed to gain insight into the tumor suppressor gene Hint1. A major reprogramming of lipid homeostasis was found in both serum and liver of Hint1-null (Hint(-/-)) mice, with significant changes in the levels of many lipid molecules, as compared with gender-, age-, and strain-matched WT mice. In the Hint1(-/-) mice, serum total and esterified cholesterol were reduced 2.5-fold, and lysophosphatidylcholines (LPCs) and lysophosphatidic acids were 10-fold elevated in serum, with a corresponding fall in phosphatidylcholines (PCs). In the liver, MUFAs and PUFAs, including arachidonic acid (AA) and its metabolic precursors, were also raised, as was mRNA encoding enzymes involved in AA de novo synthesis. There was also a significant 50% increase in hepatic macrophages in the Hint1(-/-) mice. Several hepatic ceramides and acylcarnitines were decreased in the livers of Hint1(-/-) mice. The changes in serum LPCs and PCs were neither related to hepatic phospholipase A2 activity nor to mRNAs encoding lysophosphatidylcholine acetyltransferases 1-4. The lipidomic phenotype of the Hint1(-/-) mouse revealed decreased inflammatory eicosanoids with elevated proliferative mediators that, combined with decreased ceramide apoptosis signaling molecules, may contribute to the tumor suppressor activity of Hint1.

Keywords: apoptosis; carcinogenesis; cholesterol; eicosanoids; lipidomics; mass spectrometry; metabolomics; phospholipids; proliferation.

Fig. 1.

Metabolomic and lipidomic analysis of serum and liver extracts in Hint1^−/− (KO) and WT mice. A: Serum metabolomics, with the PCA scores plots and OPLS-DA loadings S-plots for ESI+ and ESI− modes. B: Serum lipidomics, with PCA scores plots and OPLS-DA loadings S-plots for ESI+ and ESI− modes. C: Liver lipidomics, with PCA scores plots and OPLS-DA loadings S-plots for ESI+ and ESI− modes. Green and red symbols represent WT and KO mice, respectively. In the OPLS-DA loadings S-plots, red, blue, green, orange, and purple symbols represent PC, CER, SM, DG, and LPC, respectively.

Fig. 2.

Quantitation of lipids in serum of Hint1^−/− (KO) and WT mice. A: Rise in serum LPC concentrations (μM), fall in serum PC concentrations and rise in serum LPA concentrations in KO mice compared with WT mice. B: Changes in hepatic phospholipids (nmol/mg wet liver) between KO mice and WT mice. C: Changes in ceramide concentrations (units/mg wet liver) between KO mice and WT mice. Analyses were performed in duplicate for WT (n = 6) and KO (n = 6) mice. * P < 0.05; ** P < 0.001; *** P < 0.0001.

Fig. 3.

Quantitation of fatty acids in liver of Hint1^−/− (KO) and WT mice. A: No change in saturated fatty acids 16:0 and 18:0, but highly statistically significant elevations in monounsaturated (18:1) and polyunsaturated (20:5 and 22:6) essential fatty acids in KO mice compared with WT mice. Analyses were performed in duplicate for WT (n = 6) and KO (n = 6) mice. B: De novo synthesis of arachidonic acid from α-linoleic acid by desaturation and elongation in liver of KO and WT mice, showing the three enzymes involved and the fold change in mRNA expression for each of these enzymes determined by QPCR. AA and precursors are synthesized more in KO liver than in WT liver, consistent with the gene expression results.

Fig. 4.

Histopathology and immunohistochemistry for signs of hepatic inflammation. A: Detection of macrophages in a WT formalin-fixed liver section. Brown staining indicates macrophages. B: Detection of macrophages in a Hint1^−/− formalin-fixed liver section. Brown staining indicates macrophages. C: Quantitation of lymphocyte infiltration by histopathology. D: Quantitation of F4/80 IHC for murine hepatic macrophages using three sections per liver (three sections for each of six mice per genotype). ** P < 0.001.