Figure 1.

Low B12 impedes the metformin action of lowering TG and expressing genes in fatty acid synthesis. (A) Total intracellular levels of TG quantified in cells using the TG kit (ab65336) (Abcam Plc, Cambridge, UK) and normalized per milligram protein under each B12 condition. (B) Levels of synthesized TG in cells assessed with the radiochemical flux assay. HepG2 was first labelled with ¹²C-Oleate for 2 h, then followed by total lipid extraction, and the resultant radiolabelled triglyceride was separated on a thin-layer chromatography (TLC) plate with glyceryltripalmitate as standard and quantified with the scintillation counter Beckman coulter LS6500 (Beckman Coulter, Woonsocket, RI, USA) and normalized per milligram protein estimated with the Bradford method. (C) The mRNA expression of nuclear transcription factor SREBF1 (i) and enzymes regulating de novo fatty acid synthesis [ACLY (ii), ACC (iii) FASN (iv) and ELOVL6 (v)], (D) TG synthesis such as [SCD (i), GPAM (ii), AGPAT (iii), DGAT2 (iv) and DGAT1 (v)] and (E) cholesterol [LDLR (i), HMGCR (ii) and (HMGCS1 (iii)] normalized to 18S rRNA endogenous control (Applied Biosystems, Knutsford, UK). The data is representative of mean ± SEM (n = 6), and * represents significance compared to controls B12 (500 nM) and low B12 (25 pM), $—compared to controls B12 (500 nM) in 1 mM and 2 mM metformin and &—compared to low B12 (25 pM) with 1 mM metformin and 2 mM, respectively; * p < 0.05, ** p < 0.01, *** p < 0.001; $ < 0.05, $$ < 0.01, $$$ < 0.001; & < 0.05, && < 0.01, &&& < 0.001.