Vitamin B12 Induces Hepatic Fatty Infiltration through Altered Fatty Acid Metabolism

Abstract

Background/Aims: Rise in global incidence of obesity impacts metabolic health. Evidence from human and animal models show association of vitamin B12 (B12) deficiency with elevated BMI and lipids. Human adipocytes demonstrated dysregulation of lipogenesis by low B12 via hypomethylation and altered microRNAs. It is known de novo hepatic lipogenesis plays a key role towards dyslipidaemia, however, whether low B12 affects hepatic metabolism of lipids is not explored. Methods: HepG2 was cultured in B12-deficient EMEM medium and seeded in different B12 media: 500nM(control), 1000pM(1nM), 100pM and 25pM(low) B12. Lipid droplets were examined by Oil Red O (ORO) staining using microscopy and then quantified by elution assay. Gene expression were assessed with real-time quantitative polymerase chain reaction (qRT-PCR) and intracellular triglycerides were quantified using commercial kit (Abcam, UK) and radiochemical assay. Fatty acid composition was measured by gas chromatography and mitochondrial function by seahorse XF24 flux assay. Results: HepG2 cells in low B12 had more lipid droplets that were intensely stained with ORO compared with control. The total intracellular triglyceride and incorporation of radio-labelled-fatty acid in triglyceride synthesis were increased. Expression of genes regulating fatty acid, triglyceride and cholesterol biosynthesis were upregulated. Absolute concentrations of total fatty acids, saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), trans-fatty acids and individual even-chain and odd-chain fatty acids were significantly increased. Also, low B12 impaired fatty acid oxidation and mitochondrial functional integrity in HepG2 compared with control. Conclusion: Our data provide novel evidence that low B12 increases fatty acid synthesis and levels of individual fatty acids, and decreases fatty acid oxidation and mitochondrial respiration, thus resulting in dysregulation of lipid metabolism in HepG2. This highlights the potential significance of de novo lipogenesis and warrants possible epigenetic mechanisms of low B12.

Metformin Inefficiency to Lower Lipids in Vitamin B12 Deficient HepG2 Cells Is Alleviated via Adiponectin-AMPK Axis

“…Our studies in adipocytes demonstrated B12-induced reduction in methylation potential and hypo-methylation at promoter regions of low-density lipoprotein receptor (LDLR) and sterol regulatory element binding protein (SREBF1) genes [8]. Likewise in hepatocytes, we provided evidence of low B12-induced dysregulation of intrahepatic fatty acid metabolism [9]. The risk of low B12 levels increases with higher metformin dose and longer treatment duration, as well as in patients with risk factors for vitamin B12 deficiency [10].…”

“…Cell culture: Briefly, culture of the HepG2 cell line was carried out in T75-flasks and seeded in 6-well plates with custom-made EMEM medium supplemented with different B12 concentrations, namely, 500 nM (control), 1 nM (1000 pM), 100 pM and 25 pM (low B12), as previously described [9] (details of HepG2 source, authenticity and mycoplasma assays have been provided in Supplementary Materials). After the last medium change in plates with cells at 90-100% confluence, the EMEM-B12 medium was then replaced with a serum-free medium (0.1% BSA, 1% L-Glutamine) overnight.…”

“…Total intracellular TG estimation: Estimation of total intracellular TG in the cells was performed using the commercial triglyceride quantification kit (ab65336) from Abcam, Cambridge, UK as previously described [9]. Briefly, reconstitution and preparation of the TG standard, probe, enzyme mix and lipase were carried out following the manufacturer's protocol.…”

“…The cells were harvested in 2 mL methanol, and the synthesized radiolabelled-TG was extracted using a mobile phase, such as hexane/diethyl ether/formic acid (v/v/v, 70/30/1), on a thin-layer chromatography (TLC) plate with 10 nmol glyceryltripalmitate (tripalmitin) as a standard. The TG bands on the TLC plates were transferred into vials containing 5 mL scintillation fluid for scintillation counter-assessment of radioactivity and normalized per milligram (mg) protein, as described previously [9].…”

Interaction between Metformin, Folate and Vitamin B12 and the Potential Impact on Fetal Growth and Long-Term Metabolic Health in Diabetic Pregnancies

“…Emerging studies suggest that another potential mechanism by which metformin-induced impairment of one carbon metabolism may influence events in the placenta and fetus is via regulation of mitochondrial function. Yang et al 2020 reported a new role for one carbon metabolism, in which it may be linked to mitochondrial respiration via NADH production of serine catabolism [ 18 ], whilst Boachie et al 2021 demonstrated that B 12 deficiency impaired mitochondrial respiration [ 170 ]. These findings, together with evidence of placental mitochondrial dysfunction in GDM pregnancy [ 171 ], suggest that further studies are needed to elucidate whether metformin-mediated B 12 /folate deficiency exerts a similar impact on mitochondrial function in diabetic pregnancy.…”