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. 2020 Mar 6;295(10):3330-3346.
doi: 10.1074/jbc.RA120.012533. Epub 2020 Jan 23.

Metformin Lowers Glucose 6-phosphate in Hepatocytes by Activation of Glycolysis Downstream of Glucose Phosphorylation

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Free PMC article

Metformin Lowers Glucose 6-phosphate in Hepatocytes by Activation of Glycolysis Downstream of Glucose Phosphorylation

Tabassum Moonira et al. J Biol Chem. .
Free PMC article

Abstract

The chronic effects of metformin on liver gluconeogenesis involve repression of the G6pc gene, which is regulated by the carbohydrate-response element-binding protein through raised cellular intermediates of glucose metabolism. In this study we determined the candidate mechanisms by which metformin lowers glucose 6-phosphate (G6P) in mouse and rat hepatocytes challenged with high glucose or gluconeogenic precursors. Cell metformin loads in the therapeutic range lowered cell G6P but not ATP and decreased G6pc mRNA at high glucose. The G6P lowering by metformin was mimicked by a complex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol) and by overexpression of mGPDH, which lowers glycerol 3-phosphate and G6P and also mimics the G6pc repression by metformin. In contrast, direct allosteric activators of AMPK (A-769662, 991, and C-13) had opposite effects from metformin on glycolysis, gluconeogenesis, and cell G6P. The G6P lowering by metformin, which also occurs in hepatocytes from AMPK knockout mice, is best explained by allosteric regulation of phosphofructokinase-1 and/or fructose bisphosphatase-1, as supported by increased metabolism of [3-3H]glucose relative to [2-3H]glucose; by an increase in the lactate m2/m1 isotopolog ratio from [1,2-13C2]glucose; by lowering of glycerol 3-phosphate an allosteric inhibitor of phosphofructokinase-1; and by marked G6P elevation by selective inhibition of phosphofructokinase-1; but not by a more reduced cytoplasmic NADH/NAD redox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMP-activated protein kinase-independent mechanism through changes in allosteric effectors of phosphofructokinase-1 and fructose bisphosphatase-1, including AMP, Pi, and glycerol 3-phosphate.

Keywords: glucose 6-phosphate; glycolysis; hepatocyte; liver; metformin; phosphofructokinase.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Metformin accumulation in hepatocytes and effects on cell G6P and ATP. A and B, cell metformin in mouse hepatocytes incubated in MEM with 5 mm glucose and [14C]metformin at the concentrations indicated for 2 h followed by a further hour with added 25 mm glucose (n = 4–9). Cell metformin is expressed as nmol/mg cell protein (A) or as intracellular/extracellular concentration ratio (B). C–N, rat or mouse hepatocytes were incubated for 2 h in MEM containing 5 mm glucose and the metformin concentrations indicated without (open bars) or with (shaded bars) S4048, followed by a further 1 h with the substrates indicated for determination of G6P (C–I) or ATP (J–M). Cell G6P is expressed as a percentage of control without S4048 (C–E) or a percentage of respective control with or without S4048 (F–I). ATP is expressed as nmol/mg protein. C and J, 5 mm glucose, rat hepatocytes; D and K, 25 mm glucose, rat hepatocytes; E and L, 25 mm glucose, mouse hepatocytes; H and M, 5 mm glucose + 5 mm-DHA; I and N, 5 mm glucose + 2 mm xylitol; F and G show data in D and E normalized to respective control (means ± S.E. for n = 3 (E, I, L, and N), 4 (C, D, J, and K), and 7 (H and M) hepatocyte preparations). *, p < 0.05 effect of metformin (C–N); #, p < 0.05 effect of S4048 (C–E).
Figure 2.
Figure 2.
Opposite effects of AMPK activators and metformin on glycolysis and gluconeogenesis in rat hepatocytes. Incubations with metformin or AMPK activators were for 2 h followed by 1 h of incubation with substrate as in Fig. 1. A and B, phosphorylation of ACC, acetyl-CoA carboxylase–S79 by metformin and A-769662 (A) and by the three AMPK activators: C13, C-991, and A-769662 (B). Representative blots and densitometry are shown. C–E, cell G6P and ATP and production of lactate + pyruvate with 25 mm glucose. F and G, cell G6P and ATP with 5 mm DHA and 5 mm glucose. H, glucose production from 5 mm DHA in glucose-free medium. I–K, Gck, G6pc, and Pklr mRNA in rat hepatocytes after 4 h of incubation with the additions indicated at 5 or 45 mm glucose. The values are the means ± S.E. for n = 4–6 (A and B), 4–10 (C–H), and 4–5 (I–K). *, p < 0.05 relative to respective control (C–H) or relative to high glucose control (I–K).
Figure 3.
Figure 3.
Metformin lowers G6P in hepatocytes from AMPK-KO mice. A, immunoactivity to AMPK in hepatocytes from AMPKα1α2lox/lox mice (M1, M7, and M8) and AMPKα1α2lox/lox Alb-CRE (M2–M6) mice designated AMPK-KO. B, immunoactivity to AMPK-T172(P) after 3 h of incubation with metformin (0.2 or 0.5 mm) or A-769662 (10 μm) in hepatocytes from AMPKα1α2lox/lox control and AMPK-KO mice. C, immunoactivity to AMPK-T172(P) in hepatocytes from AMPKα1α2lox/lox incubated for 3 h with or without metformin (0.2 or 0.5 mm) or A-769667 (10 μm) at either 5 or 25 mm glucose + S4048, representative immunoblot and densitometry for n = 3 mice. *, p < 0.05 versus respective control; #, versus respective 5 mm glucose. D–O, hepatocytes from AMPKlox/lox (n = 3) or AMPK-KO (n = 5) mice were preincubated for 2 h with or without metformin (0.2 or 0.5 mm) or A-769662 (10 μm) for 2 h followed by 1 h of incubation in medium with either 25 mm glucose with or without S4048 or with 5 mm DHA with or without S4048 for determination of cell ATP (D–G) and G6P (H–K) expressed as nmol/mg protein. L and M, ATP from treatments with 25 mm glucose + S4048 expressed as a percentage of controls without S4048. N and O, G6P from treatments with 25 mm glucose + S4048 expressed as percentages of control with S4048. *, p < 0.05 versus respective control; #, versus substrate control without S4048 (S). Con, control.
Figure 4.
Figure 4.
Rotenone, dinitrophenol, and rhein mimic the G6P lowering by metformin. A, target sites of mitochondrial inhibitors: rotenone, complex 1 (C1); DNP, uncoupler (dissipation of proton gradient); and rhein, inhibitor of NNT. B–K, incubations with metformin and mitochondrial inhibitors were for 2 h followed by 1 h of incubation with substrate as in Fig. 1. B and C, cell G6P and ATP in rat hepatocytes incubated with mitochondrial inhibitors. The values are means ± S.E. for n = 3–15. D, glucose oxidation in mouse hepatocytes incubated with 15 mm [U-14C]glucose for 1 h (after 2 h with or without metformin). The values are means ± S.E. for n = 3. E, rhein raises cell NADP and lowers G6P (n = 5 G6P,ATP; 2 NADP). F–H, effects of metformin and 2 mm NH4Cl on NADP, G6P, and ATP (n = 4). I–K, metformin lowers G6P in hepatocytes from mice with either an intact (WT) or lacking a functional Nnt gene (Nnt del; n = 7–8; J and K). The values are means ± S.E. *, p < 0.05 versus respective control (B–K); #, p < 0.05, 25 mm versus 5 mm glucose (F).
Figure 5.
Figure 5.
G6P lowering by metformin is not explained by inhibition of glucose phosphorylation or stimulation of glycogen storage. A, lowering of G6P can occur by inhibition of G6P producing or stimulation of G6P-consuming pathways. B–K, incubations with metformin and activators or inhibitors were for 2 h followed by 1 h of incubation with substrate and radiolabel. B, metabolism of [2-3H]glucose in rat hepatocytes (n = 7), except 2.5 mm metformin (n = 2). C, metabolism of [2-3H]glucose and [3-3H]glucose in mouse hepatocytes (n = 4–5). D, glucokinase translocation 25 mm versus 5 mm glucose (n = 20–30 fields in 2–3 rat hepatocyte preparations) and representative images above. Scale bars, 10 μm. E–G, mouse hepatocytes: effects of metformin (0.2 mm) in combination with a GKA (10 μm Ro-20-1675) or mannoheptulose (MH, 10 mm) on metabolism of [2-3H],[3-3H]glucose (E), lactate and pyruvate (F), and cell G6P (G) (n = 3–9). H–J, glycogen synthesis and cell G6P: concentration-dependent inhibition of glycogen synthesis by metformin and correlation with G6P (n = 3). K, metformin inhibits glycogen storage with high glucose or DHA (n = 3). *, p < 0.05 effect of metformin or AMPK activator; #, p < 0.05 [3-3H]glucose versus [2-3H]glucose (C) or 25 mm glucose versus 5 mm glucose (D).
Figure 6.
Figure 6.
Metformin but not AMPK activators stimulates glycolysis. A, metabolism of [1,2-13C2]glucose by glycolysis (to m2, m0 lactate) and pentose pathway (m1, m0 lactate). B and C, rat hepatocytes were incubated for 1 h in MEM with 2 μm S4048, 0.4 mm α-cyanocinnamate, 0.5 mm AOA, and [1,2-13C2]glucose (15 mm) for determination of lactate m0, m1, and m2 mass isotopologs. Metformin and A-769662 were present during the final 1 h of incubation and during a 2-h preincubation. NH4Cl (2 mm) was present during the final 1 h of incubation. The values are means ± S.E. for n = 4 hepatocyte preparations. *, p < 0.05 relative to control.
Figure 7.
Figure 7.
Changes in hepatocyte G6P during selective targeting of PFK1 and/or FBP1. A and B, mouse hepatocytes were either untreated or treated with an adenoviral vector (PFK-KD) to deplete fructose 2,6-P2 and after overnight culture were incubated for 2 h with S4048 and without or with 0.2 mm metformin and for a further 1 h with 25 mm glucose. A, lactate + pyruvate production. B, cell G6P (n = 7–10). C and D, mouse hepatocytes were incubated with S4048 and the concentrations of ATA indicated for 2 h and then for 1 h with 25 mm glucose. C, cell G6P and ATP and lactate + pyruvate. D, metabolism of [2-3H]glucose and [3-3H]glucose. E, incubations were as for C but with the FBP1 inhibitor (FBPi) concentrations as indicated. F and G, mouse hepatocytes were incubated for 2 h without or with 200 μm metformin and then for a further 1 h with 25 mm glucose without or with 100 μm AOA (n = 5–6). H, cell Pi in mouse hepatocytes (n = 3) after 2 h of incubation with metformin and inhibitors and for a further 1 h with 25 mm glucose.*, p < 0.05 effect of metformin or inhibitor; #, p < 0.05 effect of PFK-KD.
Figure 8.
Figure 8.
Overexpression of mGPDH lowers G6P and mimics the metformin repression of G6pc. A–C, after cell attachment mouse hepatocytes were either untreated or treated (4 h) with an adenoviral vector (Ad-m-Gpd2 at 4.8 × 107 plaque-forming units/ml) for overexpression of mGPDH (mGPDH-OE). After overnight culture, the hepatocytes were incubated for 2 h in MEM without or with 100 μm metformin (as indicated). A and B, the medium was then supplemented with 25 mm glucose (25G), 5 mm DHA, or 2 mm glycerol, and incubations were for a further 1 h for determination of cell G3P and G6P. C, the substrate was 25 mm glucose with additional controls at 5 mm glucose, and incubations were for a further 4 h for RNA extraction and mRNA analysis, which was expressed relative to 5 mm glucose control (1.0). The values are means ± S.E. (n = 3–5). *, p < 0.05 relative to untreated. D–I, after overnight culture, mouse hepatocytes were incubated with either 25 mm glucose (25G) without or with S4048 (25G+S) or with 2 mm xylitol (Xyl) or 0.2 mm AOA alone or in combination. Incubations were either for 60 min for determination of cell G6P and G3P (D and E) or for 4 h (F–I) for RNA extraction and analysis of ChREBP-α (F), ChREBP-β (G), G6pc (H), and Gpd2 (I) mRNA, which is expressed relative to respective control at 5 mm glucose. The values are means ± S.E. (n = 4–5 (D and E) or n = 6–7 (G–I)). *, p < 0.05 versus control.
Figure 9.
Figure 9.
Effects of amino-oxyacetate and mitochondrial inhibitors on hepatocyte G3P and ATP in mouse hepatocytes. A, cell G3P represents the balance between formation from exogenous glycerol; cGPDH, which catalyzes the reversible NADH/NAD-dependent interconversion of DHAP and G3P; and mGPDH, which catalyzes the irreversible oxidation of G3P to DHAP with transfer of electrons to mitochondrial ubiquinone (Q). Rotenone inhibits the transfer of electrons from complex 1 (C1) to ubiquinone. B–O, hepatocytes were incubated for 1 h in MEM with the substrates indicated without (open bars) or with (filled bars) 200 μm amino-oxyacetate for analysis of cell G3P and ATP (nmol/mg protein). In J and K, lactate and pyruvate were determined in the medium of the 60-min incubation. The glucose (G) concentration was 5 mm unless otherwise indicated, and ethanol (L–O) was 15 mm. Metformin (100 or 200 μm), where indicated (D–G and L–O), was present in a 2-h preincubation and during the final 1-h incubation. Concentrations of rotenone (Rot), DNP, and metformin (Met) are shown in μm. The values are means ± S.E. (n = 4 (B, C, F, and G), n = 6 (D and E), and n = 2 (H–O) hepatocyte preparations each with triplicate incubations). *, p < 0.05 relative to respective control; #, p < 0.05 effect of AOA.

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