The anti-diabetic drug, metformin, is associated with progressive decrease in serum vitamin B12 levels whereas vitamin B12 deficiency is related to increased insulin resistance and dyslipidaemia by altered methylation. As the risk of vitamin B12 deficiency is increased among metformin users, it is of utmost importance to examine how metformin response in vitamin B12 deficient population.
We investigated the cellular mechanism of metformin in vitamin B12 insufficient human hepatocellular cell line (HepG2) and examined the efficacy of metformin response in vitamin B12 deficient (<150 pmol/l) gestational diabetes mellitus (GDM). HepG2 was cultured in different vitamin B12 conditions (0, 10, 100, 1000 nM) for 24 days (passaged on every 6th day for four passages). Then, they were treated with insulin 100 nM for 15 min or with metformin 2 mM for 24 h. Protein and RNA extracts were quantified for insulin signaling molecules and AMP-activated protein kinase (AMPK) and its downstream signals in lipid metabolism.
In HepG2 culture, decreased phosphorylation of insulin signaling molecules (Akt, GSK) were observed across decreasing B12 conditions. In metformin-treated hepG2 cells, Thr-172 phosphorylation of AMPK and Ser-79 phosphorylation of acetyl-CoA carboxylase (ACC) were increased in 1000 nM B12 compared to 0 nM B12 condition. Similarly, there was decreased gene expression levels of Fatty Acid Synthase (FAS) and 3-Hydroxy 3-Methylglutaryl CoA Reductase (HMGCR) enzymes in 1000 nM B12 compared to 0 nM B12 condition. There is no significant difference in the risk of metformin failure (metformin-treated GDM with additional insulin) between vitamin B12 deficient and normal GDM (odds ratio 0.91 (95% CI 0.42,2.00)).
Our preliminary results indicate that in hepatic cells differentiated in low B12 conditions, metformin phosphorylation on AMPK and its downstream molecules are reduced. This noble finding suggests the future exploration of mitochondrial function in B12 deficient cell and highlights the importance of vitamin B12 sufficiency for full potency of metformin. It is also suggested that more precise definition of metformin failure should be used for clinical studies.