Endocrine Abstracts

Background: Metformin is utilized in clinical trials for treatment of non-alcoholic fatty liver disease (NAFLD) and obesity. Metformin increases AMP:ATP ratio activating AMP activated protein kinase-alpha (AMPKα), a mediator of fatty acid oxidation (FAO) in the liver. Meanwhile, prolonged oral metformin therapy in T2DM decreases vitamin B12(B12) in patients and evidence shows that low B12 dysregulates lipid levels. We therefore investigated whether low B12 impairs FAO induced by metformin in the liver.

Methods: Liver cell line Hep G2 was cultured in custom-made B12 deficient Eagle’s Minimal Essential Medium (EMEM) and seeded in different concentrations of B12 media 500 nM (control), 1000 pM, 100 pM and 25 pM (low) B12. Hepatocytes were exposed to 24 hour treatment with 1 mM and 2 mM metformin before harvest. Gene expression, protein levels and oxygen consumption rate (OCR) were measured using real time PCR (qRT-PCR), western blotting and seahorse flux XF24.

Results: Activation of pAMPKα and pACC were decreased by low B12 (25pM). Administration of 1 mM and 2 mM metformin to low B12 hepatocytes significantly impaired the upregulation of pAMPKα and pACC, resulting in high acetyl CoA carboxylase (ACC) expression. Restoration of the rate-limiting enzyme [carnitine palmitoyl transferase 1 alpha (CPT1α)] and the downstream genes involved in FAO [carnitine acyl carnitine translocase (CACT), Long chain Acyl–CoA dehydrogenase (ACADL), Medium chain Acyl–CoA dehydrogenase (ACADM), Short chain Acyl–CoA dehydrogenase (ACADS) and long-chain 3-hydroxyacyl-CoA dehydrogenase (HADHA)] were decreased in low B12 hepatocytes treated with metformin. Finally, spare respiratory capacity was impaired in low B12 following palmitate and metformin administration.

Conclusion: B12 deficiency (1) lowers levels of pAMPKα and pACC, and (2) metformin administration in low B12 failed to restore pAMPKα and pACC, and FAO genes. Hepatocytes’ mitochondrial function was hampered by low B12 and therefore lipid lowering effect of metformin is compromised, inducing FA accumulation in the low B12 hepatocytes.