Excess S-adenosylmethionine reroutes phosphatidylethanolamine towards phosphatidylcholine and triglyceride synthesis

Abstract

Methionine adenosyltransferase 1A (MAT1A) and glycine N-methyltransferase (GNMT) are the primary genes involved in hepatic S-adenosylmethionine (SAMe) synthesis and degradation, respectively. Mat1a ablation in mice induces a decrease in hepatic SAMe, activation of lipogenesis, inhibition of triglyceride (TG) release, and steatosis. Gnmt-deficient mice, despite showing a large increase in hepatic SAMe, also develop steatosis. We hypothesized that as an adaptive response to hepatic SAMe accumulation, phosphatidylcholine (PC) synthesis by way of the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway is stimulated in Gnmt(-/-) mice. We also propose that the excess PC thus generated is catabolized, leading to TG synthesis and steatosis by way of diglyceride (DG) generation. We observed that Gnmt(-/-) mice present with normal hepatic lipogenesis and increased TG release. We also observed that the flux from PE to PC is stimulated in the liver of Gnmt(-/-) mice and that this results in a reduction in PE content and a marked increase in DG and TG. Conversely, reduction of hepatic SAMe following the administration of a methionine-deficient diet reverted the flux from PE to PC of Gnmt(-/-) mice to that of wildtype animals and normalized DG and TG content preventing the development of steatosis. Gnmt(-/-) mice with an additional deletion of perilipin2, the predominant lipid droplet protein, maintain high SAMe levels, with a concurrent increased flux from PE to PC, but do not develop liver steatosis.

Conclusion: These findings indicate that excess SAMe reroutes PE towards PC and TG synthesis and lipid sequestration.

Figure 1. Schematic representation of the role of SAMe in mediating TG synthesis via PEMT

PE, phosphatidylethanolamine; PC, phosphatidylcholine; PA, phosphatidic acid; CER, ceramide; SM, sphingomyelin; DG, diglycerides; FA, fatty acids; TG, triglycerides; LD, lipid droplets; PEMT, PE N-methyltransferase; PLD, phospholipase D; PAP, PA phosphatase; PLC, phospholipase C; SMS, sphingomyelin synthase; PLIN2, perilipin2

Figure 2. Gnmt ablation inhibits lipogenesis and enhances TG secretion

3-month-old wild type (WT) and Gnmt^−/− (KO) were fasted 2 hours before experiments were performed. (a) Hepatocytes were isolated and incubated with [³H]acetate for the time indicated. Radioactivity incorporated into TG was assessed with a scintillation counter. (b) Hepatic TG secretion rate was measured after inhibition of VLDL metabolism with 1 g/kg poloxamer (P-407). (c) Serum was isolated and ketone bodies were quantified. (d) Hepatocytes were incubated with [³H]oleate for the time indicated, the medium was harvested and the radiolabel in acid-soluble metabolites measured. (e) Quantitative RT-PCR analysis of hepatic genes in WT and KO mice. Acaca, acetyl-CoA carboxylase; Cs, citrate synthase; Fasn, fatty acid synthase; G6pdx, glucose-6-phosphate dehydrogenase; Me1, malic enzyme 1; Pygl, glycogen phosphorylase; Srebf, sterol regulatory element-binding proteins. Values are means±SEM of 4–6 animals per group. Statistical differences between KO and WT mice are denoted by *p<0.05 and **p<0.01 (Student’s t test) and by two-way ANOVA.

Figure 3. Restoration of normal hepatic flux from PE to PC in Gnmt^−/− mice after feeding a methionine deficient diet

3-month-old wild type (WT), Gnmt^−/− (KO), and Gnmt^−/− mice fed a MDD (KO-MDD) for 3 weeks were fasted 2 hours before experiments were performed. (a) Hepatocytes were isolated and incubated with [³H]ethanolamine for 4 hours, and the radioactivity incorporated into PC was expressed as a percentage of the radiolabel incorporated into PC plus PE. Microsomes were isolated from WT and Gnmt^−/− mice liver, and PE (b) and PC (c) levels quantified after lipid extraction and separation by TLC. (d) Liver PE and (e) PC were quantified as mentioned before. (f) Liver PC/PE ratio. Values are means±SEM of 4–6 animals per group. Statistical differences between Gnmt^−/− and WT mice are denoted by *p<0.05 and ***p<0.001 (Student’s t test); between Gnmt^−/− and Gnmt^−/− fed a MDD are denoted by ### p<0.001 (Student’s t test).

Figure 4. Restoration of normal hepatic DG and TG content in Gnmt^−/− mice after feeding a methionine deficient diet

3-month-old wild type (WT), Gnmt^−/− (KO), and Gnmt^−/− mice fed a MDD (KO-MDD) for 3 weeks were fasted 2 hours before experiments were performed. Liver DG (a) and TG (b) were quantified after extraction and separation of lipids by TLC. (c) Hepatocytes were isolated and incubated with 3-deazaadenosine (DZA, 10 µM) for 4 hours and at the end of this period the content of TG quantified. (d) Hepatocytes were isolated and incubated with [³H]oleate for the time indicated. Radioactivity incorporated into DG was assessed with a scintillation counter. (e) Quantitative RT-PCR analysis of genes in livers of WT, Gnmt^−/− (KO), and MDD-treated Gnmt^−/− (KO-MDD) mice. Cidec, cell-death-inducing DFFA-like effector c; Fitm1, fat storage-inducing transmembrane protein 1; G0s2, G0/G1 switch gene; Plin2, perilipin 2; Pparg, peroxisome proliferator-activated receptor γ; Scd1, stearoyl-CoA desaturase 1. Values are means±SEM of 4–6 animals per group. Statistical differences between Gnmt^−/− and WT mice are denoted by **p<0.01 and ***p<0.001 (Student’s t test); between Gnmt^−/− and Gnmt^−/− fed a MDD are denoted by ### p<0.001 (Student’s t test). Multiple comparisons among groups were statistically evaluated by two-way ANOVA.

Figure 5. Deletion of PLIN2 in Gnmt^−/− mice prevents liver steatosis

3-month-old Gnmt^−/−, Gnmt^−/−/Plin2^−/− and their wild types (WT) were fasted 2 hours before experiments were performed. (a) PE, (b) PC, (c) DG, and (d) TG levels were quantified in liver after extraction and separation of lipids by TLC. (e) Liver PC/PE ratio. (f) Representative hematoxylin and eosin staining from 3-month-old wild type (WT), Gnmt^−/− and Gnmt^−/−/Plin2^−/− mice. Values are means±SEM of 4–6 animals per group. Statistical differences between Gnmt^−/− and WT mice are denoted by *p<0.05; **p<0.01; ***p<0.001 (Student’s t test).

Figure 6. Deletion of Plin2 in Gnmt^−/− mice promotes gluconeogenesis

3-month-old wild type (WT) and Gnmt^−/−/Plin2^−/− (KO) mice were fasted 2 hours before experiments were performed. (a) Hepatocytes were isolated and incubated with [³H]acetate for the time indicated. Radioactivity incorporated into TG was assessed with a scintillation counter. (b) Hepatocytes were incubated with [³H]oleate for the time indicated, the medium was harvested and the radiolabel in acid-soluble metabolites measured. (c) Serum was isolated and ketone bodies were quantified. (d) Hepatocytes were incubated in the absence or presence of lactate plus pyruvate (Lac/Pyr) at 30 mM/3 mM or 25 mM glycerol, and glucose production determined. (e) Hepatic TG secretion rate was measured after inhibition of VLDL metabolism with 1 g/kg poloxamer (P-407). Values are means±SEM of 4–6 animals per group. Statistical differences between KO and WT mice are denoted by *p<0.05, **p<0.01 and ***p<0.001 (Student’s t test) and by two-way ANOVA.

Figure 7. Identification of SAMe regulated lipids

Heat map representation of the liver lipidomic signatures obtained from Gnmt^−/−, MDD-treated Gnmt^−/−, and Gnmt^−/−/Plin2^−/− mice. Metabolite abundance ratios in liver samples comparing (a) Gnmt^−/−/Plin2^−/−, (b) Gnmt^−/−, and (c) Gnmt^−/−-MDD with their respective WT, is shown. For each comparison, log transformed metabolite abundance ratios are depicted, as represented by the scale (d), where pronounced colours correspond to significant fold-changes (p<0.05, Student¥s or Welch’s t test analysis considering 6 animals per group). Lipidomics analysis was performed as described in (20). Individual metabolite details, including fold change and statistical significance, are shown in Supplementary Table 1. TG, triglycerides; DG, diglycerides; UFA, unsaturated fatty acids; SL, sphingolipids; PE, phosphatidylethanolamine and lysoPE; PC, phosphatidylcholines and lyso PC; PI, phosphatidylinositols and lysoPI; ChoE, cholesteryl esters.