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

Figures

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  Figure 1. Low B12 impedes the metformin action of lowering TG and expressing genes in fatty acid synthesis. (A) Total intracellular levels of TG quantified in cells using the TG kit (ab65336) (Abcam Plc, Cambridge, UK) and normalized per milligram protein under each B12 condition. (B) Levels of synthesized TG in cells assessed with the radiochemical flux assay. HepG2 was first labelled with 12C-Oleate for 2 h, then followed by total lipid extraction, and the resultant radiolabelled triglyceride was separated on a thin-layer chromatography (TLC) plate with glyceryltripalmitate as standard and quantified with the scintillation counter Beckman coulter LS6500 (Beckman Coulter, Woonsocket, RI, USA) and normalized per milligram protein estimated with the Bradford method. (C) The mRNA expression of nuclear transcription factor SREBF1 (i) and enzymes regulating de novo fatty acid synthesis [ACLY (ii), ACC (iii) FASN (iv) and ELOVL6 (v)], (D) TG synthesis such as [SCD (i), GPAM (ii), AGPAT (iii), DGAT2 (iv) and DGAT1 (v)] and (E) cholesterol [LDLR (i), HMGCR (ii) and (HMGCS1 (iii)] normalized to 18S rRNA endogenous control (Applied Biosystems, Knutsford, UK). The data is representative of mean ±…

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  Figure 2. Low B12 impedes metformin action on fatty acid oxidation (FAO) and mitochondrial function via impaired AMPK: The mRNA expression of (A) fatty acid oxidation genes such as CPT1 (i), CACT (ii), HADHA (iii), ACADS (iv), ACADM (v) and ACADL (vi) normalized to 18S rRNA endogenous control (Applied Biosystems, Knutsford, UK). (B) The spare respiratory capacity of hepatocytes in: (i) substrate-rich KHB substrate medium containing glucose (2.5 mM), pyruvate (1 mM), an amino acid (L-Glutamine) (2 mM) and BSA (0.1%) at pH 7.4 under various B12 conditions; (ii) in metformin-treated hepatocytes under various B12 conditions and (iii) in a limited-substrate KHB medium containing only 0.5 mM L-carnitine and 1.25 mM glucose and supplemented with palmitate (200 µM)/BSA (33.3 µM) (basal control) under different B12 conditions. (C) Represents pAMKα (i) and pACC (ii) levels normalized to total AMPK and ACC, respectively. (D) Metformin treatment failed to restore pAMPKα (i) and pACC (ii) levels with low B12. The data is representative of mean ± SEM (n = 6), and * represents significance compared to controls B12 (500 nM) and low B12 (25 pM), $—compared to controls B12 (500 nM) in 1 mM and 2 mM…

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  Figure 3. Adiponectin ameliorates the regulation of FA synthesis in low-B12 hepatocytes via increased AMPK activation and AdipoR1/R2 (A) The mRNA expression of AdipoR1 (i) and AdipoR2 (ii) under low B12 conditions and (B) mRNA expression of AdipoR1 (i) and AdipoR2 (ii) following 24 h treatment with 25 ng adiponectin. (C) Protein levels of pAMPKα (i) and pACC (ii) normalized to total AMPK and ACC, respectively in adiponectin [or AICAR (positive control)] treated hepatocytes. (D) mRNA expression of enzymes nuclear transcription factor SREBF1 (i) and enzymes regulating FA synthesis, such as FASN (ii), ACC (iii), ELOVL6 (iv); (E) triglyceride synthesis, such as SCD (i), DGAT1 (ii) and DGAT2 (iii); and (F) cholesterol synthesis, such as LDLR normalized to 18S rRNA endogenous control (Applied Biosystems, Knutsford, UK). (G) Total intracellular levels of triglycerides quantified in hepatocytes using the TG kit (ab65336) (Abcam Plc, Cambridge, UK) and normalized per milligram protein under each B12 condition. (H) Levels of synthesized TG in hepatocytes assessed with the radiochemical assay and quantified as disintegrations per minute (DPM) using the scintillation counter and normalized per…

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  Figure 4. Adiponectin ameliorates the regulation of FAO in low-B12 hepatocytes via increased AMPK activation: (A) The mRNA expression of enzymes regulating FAO pathway, such as CPT1a (i) and ACADM (ii), were normalized to 18S rRNA endogenous control (Applied Biosystems, Knutsford, UK). (B) (i) Oxygen consumption rate (OCR) and (ii) spare respiratory capacity following treatment with 25ng adiponectin (or 1 mM AICAR, positive control) in hepatocytes incubated with a rich-substrate KHB medium containing glucose (2.5 mM), pyruvate (1 mM), an amino acid (L-Glutamine) (2 mM) and BSA (0.1%) at pH 7.4 under various B12 conditions. (C) (i) Oxygen consumption rate (OCR) and (ii) spare respiratory capacity following injection with palmitate (200 µM)/BSA (33.3 µM) (basal control) in 25 ng adiponectin (or 1 mM AICAR, positive control)-treated-hepatocytes in a limited-substrate KHB medium containing only 0.5 mM L-carnitine and 1.25 mM glucose under different B12 conditions. (D). (i). Oxygen consumption rate (OCR) and (ii) spare respiratory capacity in hepatocytes exposed to palmitate (200 µM) and respiratory inhibitors (Oligomycin, FCCP and rotenone/antimycin). The data is representative of mean ± SEM…

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  Figure 5. Combined treatment of adiponectin and metformin ameliorates the regulation of FA synthesis in low-B12 hepatocytes via increased AMPK activation: (A) The mRNA expression of AdipoR1 (i) and R2 (ii) and master regulator of lipogenesis SREBF1 (C) (i) as well as enzymes regulating fatty acid synthesis, including ACC (ii), FASN (iii) and ELOVL6 (iv), TG synthesis (D), including SCD (i), DGAT1 (ii) and DGAT2 (iii) and cholesterol LDLR (E), were normalized to 18S rRNA endogenous control (Applied Biosystems, Knutsford, UK). (B) Protein levels of pAMPKα (i) and pACC (ii) following combined or sequential treatment of adiponectin and metformin and normalized to total AMPK and ACC, respectively. (F) Total intracellular levels of triglycerides quantified in hepatocytes using the TG kit (ab65336) (Abcam Plc, Cambridge, UK) and normalized per milligram protein under each B12 condition. (G) Levels of synthesized TGs in hepatocytes assessed with the radioactive flux assay and quantified as disintegrations per minute (DPM) using the scintillation counter and normalized per milligram (mg) protein. The data is representative of mean ± SEM (n = 6), and * represents significance compared to controls…

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  Figure 6. Combined treatment of adiponectin and metformin ameliorates the regulation of FAO in low-B12 hepatocytes via increased AMPK activation: The mRNA expression of key enzymes regulating FAO (A) such as CPT1 (i) and ACADM (ii) normalized to 18S rRNA endogenous control (Applied Biosystems, Knutsford, UK). (B) (i) Oxygen consumption rate (OCR) and (ii) spare respiratory capacity following combined treatment with 25 ng adiponectin and 2 mM metformin in hepatocytes incubated with a rich-substrate KHB medium containing glucose (2.5 mM), pyruvate (1 mM), amino acid (L-Glutamine) (2 mM) and BSA (0.1%) at pH 7.4 under various B12 conditions. (C) (i) Oxygen consumption rate (OCR) and (ii) spare respiratory capacity following injection with palmitate (200 µM)/BSA (33.3 µM) (basal control) in adiponectin and metformin-treated hepatocytes in a limited-substrate KHB medium containing only 0.5 mM L-carnitine and 1.25 mM glucose under different B12 conditions. (D) (i) Oxygen consumption rate (OCR) and (ii) spare respiratory capacity in hepatocytes exposed to palmitate (200 µM) and respiratory inhibitors (Oligomycin, FCCP and rotenone/antimycin). The data is representative of mean ± SEM (n = 6), and…

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  Figure 7. Schematic representation of hepatocytes with low B12 status upon metformin and adiponectin treatment: (A) Following metformin treatment (dotted line—less activation) with low B12, pAMPKa and pACC levels are decreased with subsequent increase in the levels of SREBF and malonyl-CoA that further inhibits CPT1. In effect, lipogenesis is upregulated whereas fatty acid oxidation (FAO) is decreased, resulting in accumulation of lipids in the hepatocytes. (B) Adiponectin and metformin (solid line—activation) together results in the activation of AMPK. Activated AMPK directly phosphorylates ACC which results in the inhibition of malonyl-CoA. Decreased malonyl-CoA levels facilitate the upregulation of CPT1 and subsequently improves mitochondrion function with increased oxidation of fatty acids (FAO). Similarly, activation of AMPK results in the inactivation of SREBF, which subsequently decreases lipogenesis in hepatocytes (created with BioRender.com).