Background: Metformin is currently the first drug of choice for treatment of type 2 diabetes (T2D). Metformin is known to reduce lipid levels through activation of AMP activated protein kinase-alpha (AMPKα). Metformin induces deficiency of vitamin B12 (B12) in patients with T2D. In humans, studies have shown that low B12 is associated with dyslipidemia (higher triglycerides and low HDL). Therefore, we investigated whether B12 deficiency may impair metformin action from achieving the desired lipid lowering effect in the liver.
Methods: Hep G2 cell line was cultured using custom made B12 deficient Eagles Minimal Essential Medium (EMEM) and seeded in four different concentrations of B12 media such as 500 nM (control), 1000 pM, 100 pM and 25 pM (low) B12 until 100% confluence was achieved. The cells were exposed to 24 hour treatment with 1 mM and 2 mM metformin before harvest. Protein and gene expressions were characterized using western blotting and real time PCR (qRT-PCR) respectively.
Results: Low B12 (25pM) in HepG2 cell line decreased levels of AMPKα and its downstream target pACC, compared to control. Administration of increasing concentrations of metformin (1 mM and 2 mM) to low B12 hepatocytes significantly impaired the upregulation of pAMPKα and pACC. In addition, we found that downregulation of nuclear transcriptional factor sterol regulatory element binding protein (SREBF1) and the genes involved in hepatic de novo fatty acid synthesis pathway, [fatty acid synthase (FASN), acetyl coenzyme A carboxylase (ACC) and elongation-of very-long-chain fatty acid (ELOVL6)] and TG biosynthesis [glycerol-3-phosphate acyltransferase (GPAT) and diacylglycerol acyl transferase 2 (DGAT2)] were significantly impaired in low B12 cells treated with metformin.
Conclusion: Our study provides novel evidence that Vitamin B12 deficiency (1) lowers levels of pAMPKα and pACC, and (2) metformin administration in low B12 hepatocytes failed to restore the levels of pAMPKα and pACC, and the genes involved in lipid metabolism. The mechanisms involving regulation via AMPK requires further studies.