TNF-dependent hepatic steatosis and liver degeneration caused by mutation of zebrafish s-adenosylhomocysteine hydrolase

DEV027565 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Ahcy morpholino injection leads to hepatic steatosis. Oil Red O (ORO) staining of 5-dpf larvae injected with control (A) or antisense morpholino directed against the ahcy AUG site (B). Injections were performed at 2 dpf. Note the diffuse ORO staining of the liver (outlined) in B, which is not present in the control. Injection with a splice-blocking morpholino had a similar effect.

• Supplemental Figure S2 -

  Fig. S2. Phosphatidylcholine/phosphatidylethanolamine ratio is unchanged in dtp. Total lipids were extracted from 5-dpf frozen larvae (n=20, ∼6 mg) as per standard protocol with the extraction volumes increased 10-fold to increase extraction efficiency. Reversed-phase chromatographic separation was performed on the lipid extract, with the internal standards of DMPE and DMPC (1 µM), and resolved using a multistep gradient and a C18 column (BDS Hypersil-Keystone, 2.1×250 mm column, Thermo-Fisher). The solvents used were Solvent A (50% methanol/water) and Solvent B (30:70 chloroform:methanol, 0.1% formic acid, pH 5.6). Mass spectrometric analysis of the phospholipids was performed using a triple quad mass spectrometer (QTrap 2000, Applied Biosystems) utilizing the parent-to-product ion transitions to identify phosophatidylcholine (PCr184 M+H⁺) and phosphatidyethanolamine (PE, neutral loss M-141⁺). Quantitation of the peak area of the phospholipid concentrations was relative to that of the spiked internal standards, with peak identification and quantification performed using the Analyst Classic method in Analyst 1.4 (Applied Biosystems).The ratio of PC/PE is recorded on the y-axis. Note the lack of any difference. P is not significant.

• Supplemental Figure S3 -

  Fig. S3. Persistent liver phenotype and increased tnfa expression in gnotobiotic dtp larvae. (A,B) Left lateral view of transferrin (tfa) in situ hybridizations shows reduced liver size in gnotobiotic dtp versus wild-type larvae. (C) tnfa expression remains elevated in gnotobiotic dtp larvae. (D,E) Tryptic soy agar plates plated with germ-free medium (D) or standard medium (E).

• Supplemental Figure S4 -

  Fig. S4. 5-Azacytidine treatment does not phenocopy dtp liver defects. (A,B) Lateral views of 5-dpf wild-type larvae injected with azacytidine (B) or vehicle (control) at 2, 3 and 4 dpf. (C) Reduced DNA methylation in azacytidine-injected versus control larvae as revealed by anti-methylcytosine (αMeC) slot blot of genomic DNA, with Methylene Blue counterstain (MeBl). (D,E) Comparable liver histology in 5-dpf control and azacytidine-injected larvae. (F) Liver electron micrograph from an azacytidine-injected larva shows normal mitochondrial ultrastructure and no evidence of steatosis. (G) tnfa expression is not elevated in azacytidine-injected larvae.

• Supplemental Figure S5 -

  Fig. S5. Simplified schematic of the methionine metabolism pathway used to illustrate two theories to account for altered SAM and SAH levels in adult heterozygous dtp fish and in adult wild-type fish treated with the Ahcy inhibitor 3-deaza-adenosine. (A) Reduced AHCY activity causes a modest increase in SAH that is not detected in heterozygous dtp or inhibitor-treated tissues. This modest elevation of SAH is sufficient to inhibit the activity of glycine N-methyltransferase (GNMT), an abundant methyltransferase that catalyzes the conversion of glycine to sarcosine. Inhibition of GNMT in heterozygous dtp tissue is predicted to raise SAM levels, as occurs in human and murine GNMT deficiency (Luka et al., 2006; Martinez-Chantar et al., 2008). (B) An alternative mechanism to maintain normal SAH levels in the setting of reduced AHCY activity posits increased metabolism of homocysteine to methionine, and ultimately SAM, by BHMT. Feedback inhibition by methylated DNA, RNA, protein, lipids and other compounds (i.e. sarcosine) on their respective methyltransferases sustains elevated SAM in this model, without increasing SAH levels.