Corresponding Author: Antonysunil Adaikalakoteswari and Ponnusamy Saravanan
Department of Biosciences, School of Science and Technology, Nottingham Trent University, Clifton, Nottingham NG11 8NS (UK)
Tel. Tel. +44-1158-483946, E-Mail email@example.com
Clinical Sciences Research Laboratories, University of Warwick, UHCW Campus, Clifford Bridge Road, Coventry CV2 2DX (UK)
Tel. +44-2476-968668; E-Mail P.Saravanan@warwick.ac.uk
Vitamin B12 Induces Hepatic Fatty Infiltration through Altered Fatty Acid Metabolism
Joseph Boachiea Antonysunil Adaikalakoteswaria,b Antonio Gázquezc Victor Zammita Elvira Larquéc Ponnusamy Saravanana,d,e
aDivision of Metabolic and Vascular Health, Clinical Sciences Research Laboratories, Warwick Medical School, University of Warwick, University Hospital-Walsgrave Campus, Coventry, UK, bDepartment of Bioscience, School of Science and Technology, Nottingham Trent University, Clifton, Nottingham, UK, cDepartment of Physiology, Faculty of Biology, University of Murcia, Murcia, Spain, dDiabetes Centre, George Eliot Hospital NHS Trust College Street, Nuneaton, Warwickshire, UK, ePopulations, Evidence and Technologies, Division of Health Sciences, Warwick Medical School, University of Warwick, Coventry, UK
The impact of higher adiposity on metabolic health has been extensively studied due to alarming increase in the global incidence of obesity. It was recently estimated that the worldwide prevalence of overweight in adults is 39%, obesity is 13% and non-alcoholic fatty liver disease (NAFLD) is 25% [1, 2]. Obesity, characterized by the excessive storage of fat in adipose and hepatic tissues, has been associated with dyslipidaemia, insulin resistance and NAFLD. Hepatic de novo lipogenesis is a major factor contributing to plasma triglyceride levels and hepatic steatosis . Similarly, fatty acid oxidation is significant in suppressing lipolysis of adipose triglyceride as well as reducing hepatic fat accumulation . However, increased plasma free fatty acid levels have been associated with insulin resistance, inflammation and gestational diabetes (GDM) .
Environmental factors such as nutrition (micronutrients), may affect metabolism of lipids and deregulate the processes of lipogenesis and lipid oxidation. Deficiencies in methyl donors (vitamin B12 (B12) and folate) are associated with obesity , liver steatosis  and increased risk of metabolic syndrome . Low B12 in pregnancy was associated with elevated BMI, insulin resistance and GDM . Maternal low B12 was associated with higher levels of triglyceride in both maternal and cord blood , and higher insulin resistance in the offspring at six years of age . It has also been shown that low serum B12 level was observed in individuals with NAFLD, especially with grade 2 and 3 steatosis . In support to this human studies, B12 restriction in maternal rat models demonstrated that the offspring had higher adiposity, dyslipidaemia, upregulation of enzymes in lipogenesis and lipid oxidation . We have previously shown in a human adipocyte model in low B12, the master regulator of lipogenesis (SREBF - sterol regulatory element binding protein) and cholesterologenesis (LDLR - low density lipoprotein receptor) were upregulated  and might lead to higher adiposity and dyslipidaemia via hypomethylation of DNA  and altered microRNAs . Compared to the liver, the contribution of adipose tissue to the circulating levels of lipids is considerably lower . Therefore, if the effects of B12 are similar in hepatocytes, this may explain the association observed between low B12 and dyslipidaemia in humans  and the causal association observed in animal models .
In animal models, apart from the increased triglyceride and cholesterol levels, low B12 was associated with altered levels of long chain polyunsaturated fatty acids (LC-PUFAs) in the plasma and liver of subjects across three generations . Recent evidences suggest that fatty acids in circulation may be implicated in metabolic dysregulation such as GDM , postmenopausal obese and overweight women , metabolic syndrome , type 2 diabetes (T2D)  and cardiovascular disease (CVD) . Despite the diverse metabolic roles of these fatty acids and the potential link between low B12 and beta-oxidation of fatty acids [24, 25], there are no studies that explored the relationship between low B12 on the individual fatty acids at tissue levels, especially in an active metabolic tissue such as hepatocytes.
With this foregoing discussion, in this current study we present data on (1) the effects of low B12 on lipogenesis and lipid oxidation in human hepatocyte cell line (HepG2) and (2) the effects of low B12 on the fatty acid concentrations in HepG2.
Materials and Methods
Experimental methods are detailed in Supplemental Methods (for all supplementary material see www.cellphysiolbiochem.com). They are articulated in brief in the following paragraphs.
HepG2 cell culture was done, with slight modifications . Using B12-deficient Eagles’ Minimal Essential Medium (EMEM), cells were cultured in T-75 flask and seeded into six well-plates at 75,000 cells/well in different B12 concentrations of EMEM media: 500nM (Control), 1000pM, 100pM and 25pM. Customised B12 medium was changed every 48-hours until 100% confluence.
Oil Red O staining and elution assay
HepG2 cells, following 1-hour fixation with 10% formalin, were stained for 2-hours with ORO. Oil droplet images were captured under 40x objective of light microscope.
RNA isolation, cDNA synthesis and gene expression
RNA was isolated using the Trizol method  and gene expression assays were done using qRT-PCR and normalized with 18s rRNA (Applied Biosystems, UK) .
Total intracellular triglyceride estimation
Total intracellular triglyceride in HepG2 was assessed with commercial Triglyceride Quantification Kit (ab65336) from Abcam, Cambridge, UK.
Radiochemical measurement of synthesized triglyceride
HepG2 was labelled with 12C-Oleate for 2-hours, then followed by total lipids extraction  and the resultant radiolabelled triglyceride was separated on a TLC plate with glyceryltripalmitate as standard and quantified with the scintillation counter (Beckman coulter LS6500, USA)  and normalized per milligram protein estimated with Bradford method .
Fatty acid composition in total lipids of HepG2
Fatty acid levels (µg) were normalized per milligram protein, quantified by Bradford assay , in HepG2 pellets. Pellets were dissolved in 0.2 mL cell lysis buffer containing 1mM phenylmethanesulfonyl fluoride , followed by sonication, to obtain cell lysate. Total lipids extraction  was achieved after adding 0.05mg pentadecanoic acid (internal standard) to cell pellets. After drying, synthesis of fatty acid methyl esters (FAME) using 3mol/l methanolic HCl (Supelco, Bellafonte, PA, EEUU) for 1-hour at 90°C, was performed. FAME were analysed by gas-chromatography  and fatty acid concentrations were determined in relation to peak area of internal standard.
Seahorse extracellular flux assay of mitochondrial dysfunction
Maximal respiratory capacity. Briefly, the basal oxygen consumption rate (OCR) measurement was performed in HepG2 cells in a rich substrate media (glucose-2.5mM, pyruvate-1mM, L-Glutamine-2mM, BSA-0.1%) by addition of the inhibitors Oligomycin and FCCP followed by antimycin/rotenone using Seahorse 24XF flux analyser.
Respiratory capacity in a limited-substrate (high-palmitate) supply. Then, to examine how the low B12 HepG2 function with the endogenous supply of high extracellular levels of palmitate and other limited substrate, we incubated HepG2 in a limited-substrate KHB medium (only 0.5mM L-carnitine and 1.25mM glucose), which is poorly enriched with other supplements compared with the rich-substrate KHB medium, for one-hour. After the basal OCR was measured in HepG2, the cells were either exposed to 200µM palmitate (dissolved in 33.3 µM BSA) or 33.3µM BSA only (basal control) in the substrate medium to assess how HepG2 cells efficiently uptake palmitate for ATP metabolism.
All quantitative measurements, where applicable, were obtained n=6 for standards, controls and cases for precision. Differences between either parametric groups or non-parametric groups were observed respectively by performing Student’s t-test or Mann-Whitney U test. P values of <0.05 were considered statistically significant.
Effect of B12 on Lipogenesis
Lipid droplets accumulation. To determine the effect of B12 on hepatic lipogenesis, we imaged lipid droplets in HepG2 cells using x40 objective of a light microscope under different conditions of B12 [500nM (control), 1000pM, 100pM and 25pM] following the initial fixing and staining of cells with ORO. We observed HepG2 cells in low B12 had high number of intensely stained lipid droplets compared to control with few lightly stained lipid droplets (Fig. 1A.i). Then the lipid content was quantified using the elution assay standardized by milligram protein concentration of HepG2, which showed significantly higher amount of lipids eluted from cells of low B12 compared with control (Fig. 1A.ii).
Total intracellular triglyceride levels. HepG2 in low B12 had significantly higher level of total intracellular triglyceride, normalized per milligram protein, compared with control cells (Fig. 1B), thus confirming the earlier evidence obtained in ORO staining and elution assays.
Triglyceride synthesis utilizing radio-labelled fatty acid. We observed a high measure of radioactivity (disintegration per minute, DPM) by scintillation count in HepG2 of low B12, normalized per milligram protein, following initial extraction and isolation of radiolabelled triglyceride. This provided a direct indication that increased levels of fatty acids were incorporated and synthesised in HepG2 treated with low B12 compared to control (Fig. 1C).
Genes regulating fatty acid synthesis. Next, we assessed the effect of B12 on gene expression of sterol regulatory element-binding protein (SREBF), which is a master regulator of biosynthesis pathways of fatty acids, triglyceride and cholesterol, and then the downstream genes regulating fatty acid synthesis: acetyl-CoA by ATP citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), and elongation of very long-chain fatty acids (ELOVL6). We observed that gene expression of SREBF1 and the genes involved in de novo lipogenesis including ACLY, ACC, FASN and ELOVL6, were increased in low B12 cells compared with control (Fig. 1D).
Effect of B12 on mitochondrial functional integrity
em>Efficiency of mitochondria in utilizing a rich-substrate supply. The key metabolic pathways such as fatty acid oxidation and oxidative phosphorylation (OXPHOS) in mitochondria is the principal source of energy (ATP) in eukaryotes. It has been shown that primary deficiencies in fatty acid oxidation results in secondary OXPHOS defects, although the precise underlying mechanism is unclear . We assessed the effect of B12 on mitochondrial respiration (OXPHOS), by measuring the OCR in HepG2, as an assessment of mitochondrial functional integrity under various conditions of B12.
In the presence of inhibitors (oligomycin, FCCP, rotenone), the maximal respiratory capacity, OCR of HepG2 in a rich-substrate medium (high glucose), was decreased in low B12 compared with control (Fig. 4B.i). Also, the spare respiratory capacity (SRC), a measure of the capacity of electron transport chain and substrate supply to respond to elevation in energy demand, was decreased in low B12 cells compared with control (Fig. 4B ii). This suggests that the efficiency of the mitochondria in utilising a rich-substrate for energy metabolism was compromised in low B12.
Efficiency of the mitochondria in utilizing limited-substrate (low glucose, high palmitate) supply. We observed that in low B12, upon exposure to high palmitate levels, the SRC of HepG2 cells was significantly lower compared with control (Fig. 4B.iii). This suggests that the capacity of the mitochondria to catabolise long chain fatty acid (palmitate) for energy metabolism was reduced in low B12, therefore, likely to accumulate in low B12.
We express our sincere gratitude to our sponsors: Ghana Education Trust Fund; Medical Research Council, UK; Chancellor’s scholarship for international students, University of Warwick UK; QR fund, Nottingham Trent University, UK and the APC was funded by the Open Access Fund from the Nottingham Trent University.
Conceptualization: AA and PS; Methodology: JB, AA, AG, EL and VZ.; Investigations: JB, AA, AG, EL; Writing-original draft preparation: JB and AA; Writing – Review & Editing: JB, AA, PS, AG, EL and VZ; Supervision: AA and PS. All authors contributed and approved the manuscript for submission; PS is the guarantor of this work and had full access to all the data presented in the study and takes full responsibility for the integrity and the accuracy of the data analysis.
Statement of Ethics
The authors have no ethical conflicts to disclose.
The authors have no conflicts of interest to declare.
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