Fig 2: Adiponectin ameliorates the regulation of FA synthesis in low-B12 hepatocytes via increased AMPK activation and AdipoR1/R2 (A) The mRNA expression of AdipoR1 (i) and AdipoR2 (ii) under low B12 conditions and (B) mRNA expression of AdipoR1 (i) and AdipoR2 (ii) following 24 h treatment with 25 ng adiponectin. (C) Protein levels of pAMPKα (i) and pACC (ii) normalized to total AMPK and ACC, respectively in adiponectin [or AICAR (positive control)] treated hepatocytes. (D) mRNA expression of enzymes nuclear transcription factor SREBF1 (i) and enzymes regulating FA synthesis, such as FASN (ii), ACC (iii), ELOVL6 (iv); (E) triglyceride synthesis, such as SCD (i), DGAT1 (ii) and DGAT2 (iii); and (F) cholesterol synthesis, such as LDLR normalized to 18S rRNA endogenous control (Applied Biosystems, Knutsford, UK). (G) Total intracellular levels of triglycerides quantified in hepatocytes using the TG kit (ab65336) (Abcam Plc, Cambridge, UK) and normalized per milligram protein under each B12 condition. (H) Levels of synthesized TG in hepatocytes assessed with the radiochemical assay and quantified as disintegrations per minute (DPM) using the scintillation counter and normalized per milligram (mg) protein. The data is representative of mean ± SEM (n = 6), * represents significance compared to controls B12 (500 nM) and low B12 (25 pM), $—compared to controls B12 (500 nM) with adiponectin or aicar and &—compared to low B12 (25 pM) with adiponectin or aicar, respectively; * p < 0.05, ** p < 0.01, *** p< 0.001; $ < 0.05, $$ < 0.01, $$$ < 0.001; & < 0.05, && < 0.01, &&& < 0.001.

Fig 3: 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.