Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C069

Oral Communications

Vitamin B12 deficiency inhibits the lipid lowering effect of metformin in the liver

J. Boachie1, A. Antonysunil1, J. Samavat1, P. Saravanan1,2

1. Division of Metabolic and Vascular Health, Clinical Sciences Research Laboratories, Warwick Medical School, University of Warwick, Coventry, United Kingdom. 2. Diabetes Centre, George Eliot Hospital NHS Trust College Street, Nuneaton, Warwickshire, United Kingdom, CV10 7DJ, Nuneaton, United Kingdom.

Background: Metformin is currently the first drug of choice for treatment of type 2 diabetes (T2D). In addition to improving insulin sensitivity and hyperglycaemia, metformin is also known to reduce lipid levels through activation of AMP activated protein kinase-alpha (AMPKα). It is known that metformin administration induces deficiency of vitamin B12 (B12) in patients with T2D. Observational studies in humans also show 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 Eagle's Minimal Essential Medium (EMEM) and seeded in four different concentrations of B12 media such as 500nM (control), 1000pM, 100pM and 25pM (low) B12 until 100% confluence was achieved. The cells were exposed to 24hour treatment with 1mM and 2mM metformin before harvest. Gene expression assays, protein expression and mitochondrial spare respiratory capacity were characterized using real time PCR (qRT-PCR), western blotting and seahorse XF24 assays 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 (1mM and 2mM) 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. In the fatty acid oxidation (FAO) pathway, upregulation of the rate limitting enzyme carnitine palmitoyl transferase 1 alpha (CPT1α) and downstream genes carnitine acyl carnitine translocase (CACT) and Long chain Acyl -CoA dehydrogenase (ACADL) were significantly impaired in low B12 hepatocytes. Finally, the spare respiratory capacity of mitochondria in metformin treated hepatocytes under low B12 was impaired. 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. Mitochondrial functional capability in the oxidation of fatty acid was also impaired. This supports that the lipid lowering effect of metformin in vitamin B12 deficiency is compromised. The mechanisms involving regulation via AMPK requires further studies.

Where applicable, experiments conform with Society ethical requirements