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. 2018 Sep;24(9):1384-1394.
doi: 10.1038/s41591-018-0125-4. Epub 2018 Jul 23.

Metformin Inhibits Gluconeogenesis via a Redox-Dependent Mechanism in Vivo

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Free PMC article

Metformin Inhibits Gluconeogenesis via a Redox-Dependent Mechanism in Vivo

Anila K Madiraju et al. Nat Med. .
Free PMC article

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Abstract

Metformin, the universal first-line treatment for type 2 diabetes, exerts its therapeutic glucose-lowering effects by inhibiting hepatic gluconeogenesis. However, the primary molecular mechanism of this biguanide remains unclear, though it has been suggested to act, at least partially, by mitochondrial complex I inhibition. Here we show that clinically relevant concentrations of plasma metformin achieved by acute intravenous, acute intraportal or chronic oral administration in awake normal and diabetic rats inhibit gluconeogenesis from lactate and glycerol but not from pyruvate and alanine, implicating an increased cytosolic redox state in mediating metformin's antihyperglycemic effect. All of these effects occurred independently of complex I inhibition, evidenced by unaltered hepatic energy charge and citrate synthase flux. Normalizing the cytosolic redox state by infusion of methylene blue or substrates that contribute to gluconeogenesis independently of the cytosolic redox state abrogated metformin-mediated inhibition of gluconeogenesis in vivo. Additionally, in mice expressing constitutively active acetyl-CoA carboxylase, metformin acutely decreased hepatic glucose production and increased the hepatic cytosolic redox state without altering hepatic triglyceride content or gluconeogenic enzyme expression. These studies demonstrate that metformin, at clinically relevant plasma concentrations, inhibits hepatic gluconeogenesis in a redox-dependent manner independently of reductions in citrate synthase flux, hepatic nucleotide concentrations, acetyl-CoA carboxylase activity, or gluconeogenic enzyme protein expression.

Conflict of interest statement

The authors declare no competing financial interests.

Conflict of Interests

None of the authors have any conflicts of interest related to this study.

Figures

Figure 1
Figure 1
Acute IV 50 mg kg−1 metformin treatment inhibits contributions from lactate but not alanine to hepatic gluconeogenesis in normal SD rats. (a) A schematic diagram illustrating that [3-13C]lactate will enter gluconeogenesis via lactate dehydrogenase (LDH), regulated by the cytosolic redox state, and contribute to labeling in the 1 and 6 positions of glucose as well as the 2 and 5 positions due to isomerization during the conversion of fumarate to malate. PDH, pyruvate dehydrogenase; TCA, tricarboxylic acid; PEP, phosphoenolpyruvate. (b) A schematic diagram illustrating that [3-13C]alanine is converted to pyruvate via alanine aminotransferase (ALAT) independent of redox state and will also label the 1,2,5, and 6 positions of glucose. (c) Plasma glucose levels in SD rats within 2 h after acute metformin or saline treatment and infused with either [3-13C]lactate or [3-13C]alanine tracer (d,e) The liver cytosolic redox state, as represented by the [lac]:[pyr] ratio, (d) and the liver mitochondrial redox state, as measured by the [β-OHB]:[AcAc] ratio, (e) in SD rats after acute metformin treatment and tracer infusion. (f) EGP of SD rats upon acute metformin treatment plus tracer infusion. (g) Contribution of lactate to glucose from [3-13C]lactate and contribution of alanine to glucose from [3-13C]alanine as determined by the ratio of label in the 1, 2, 5 and 6 positions of glucose in metformin-treated rat livers relative to saline-treated (metformin/saline) livers. Data are mean ± SEM (af) or mean ± SD (g), ([3-13C]lactate, saline: n = 8, metformin: n = 9; [3-13C]alanine, saline: n = 6, metformin: n = 6, biological replicates). For statistical analysis, P values were calculated by two-way ANOVA with Sidak’s multiple comparisons (af), and effect size d by Cohen’s standard (g), and NS = not significant.
Figure 2
Figure 2
Acute IV 50 mg kg−1 metformin treatment inhibits contributions from lactate but not alanine to hepatic gluconeogenesis in ZDF rats. (a) Fasting plasma glucose concentrations in ZDF rats acutely treated with metformin or saline over 2 h during [3-13C]lactate and [3-13C] alanine tracer infusions. (b) Liver [lac]:[pyr] ratio, a surrogate for the cytosolic redox state, and (c) liver [β-OHB]:[AcAc] ratio, representative of the mitochondrial redox state. (d) EGP rates in saline and metformin treated animals at 2 h post-treatment. (e) The contribution of lactate to glucose as determined by the metformin/saline ratio of the label in the 1, 2, 5 and 6 positions of glucose from [3-13C] lactate. Contribution of alanine is indicated by the metformin/saline ratio of of the label in the 1, 2, 5 and 6 positions of glucose from [3-13C]alanine. Data are mean ± SEM (a–d), or mean ± SD (e), ([3-13C]lactate, saline: n=9, metformin: n=11; [3-13C]alanine, saline: n=12, metformin: n=11, biological replicates). For statistical analysis, P values were calculated by two-way ANOVA with Sidak’s multiple comparisons (a–d), and effect size d by Cohen’s standard (d), and NS = not significant.
Figure 3
Figure 3
Acute IV 50 mg kg−1 metformin treatment decreases fasting plasma glucose and inhibits EGP in the ACC DKI mouse model by modulating redox. (a) Fasting plasma glucose concentrations in the ACC DKI mice and WT littermates before and 2 h after acute metformin treatment. (b) Hepatic cytosolic redox state and (c) liver mitochondrial redox state in ACC DKI and WT mice 2 h post-metformin or saline treatment. (d) EGP 2 h post-metformin or saline treatment in WT and ACC DKI mice. (e) Hepatic triglyceride levels in WT and ACC DKI mice 2 h post-metformin or saline treatment. (f) Liver AMPK activation in WT and ACC DKI mice as determined by the ratio of phosphorylated AMPK (P-AMPK) to total AMPK protein. (g) Liver ACC activation as determined by the ratio of phosphorylated ACC (P-ACC) to total ACC protein in ACC DKI and the WT mice. Data are mean ± SEM (WT, n=7; ACC DKI, n=9 per group for plasma glucose (a), EGP (d); n=6 per group for liver redox (b, c), TG and protein expression (e–g). For statistical analysis, P values were calculated by two-way ANOVA with Sidak’s multiple comparisons, and NS = not significant.
Figure 4
Figure 4
Infusion of substrates (pyruvate and DHA), and infusion of methylene blue (MB), both abrogate the effects of acute IV 50 mg kg−1 metformin treatment on plasma glucose and EGP in normal SD rats. (a) Fasting plasma glucose concentrations in SD rats treated acutely with saline, metformin, or metformin combined with infusion of pyruvate and DHA (both substrates that do not alter cytosolic redox state when entering gluconeogenesis) or equimolar lactate and glycerol (both substrates that increase cytosolic redox state). (b) Liver [lac]:[pyr] ratio, (c) liver [β-OHB]:[AcAc] ratio, and (d) hepatic G-3-P concentrations 2 h following acute saline, metformin, metformin with pyruvate/DHA or metformin with lactate/glycerol treatment. (e) EGP at 2 h post-treatment. (f) Fasting plasma glucose with acute saline or metformin treatment with and without concomitant treatment with 2 mg/kg acute IV MB. (g) Effect of acute saline and metformin treatments with and without MB treatment on liver cytosolic redox state and (h) mitochondrial redox state. (i) EGP 2 h post-treatment. Data are mean ± SEM, (For substrate infusion (a–e): saline, n=7; metformin, n=6; pyruvate/DHA & metformin, n=6; pyruvate/DHA & metformin, n=6; biological replicates; for MB infusion (f–i): saline, n=8; MB, n=12; metformin, n=7; MB & metformin, n=10; biological replicates). For statistical analysis, P values were calculated by one-way ANOVA with Tukey’s multiple comparisons test, and NS = not significant.
Figure 5
Figure 5
Portal vein infusion of 50 mg kg−1 metformin acutely inhibits hepatic gluconeogenesis and selectively alters lactate, β-OHB and glycerol turnover and clearance rates without altering alanine turnover and alanine clearance rates or impacting mitochondrial citrate synthase flux (VCS) in a HFD-STZ Sprague-Dawley rat model of type 2 diabetes (T2D). (a) Fasting plasma glucose concentrations in saline and metformin treated HFD-STZ rats over 2 h. (b) EGP rates in HFD-STZ rats 2 h post-metformin or saline treatment. (c) Alanine turnover and (d) clearance (clearance is mL of plasma cleared of substrate per minute) in before and after metformin treatment in HFD-STZ rats. (e) Lactate turnover, (f) lactate clearance and (g) β-OHB turnover rates in HFD-STZ rats treatment with metformin versus saline. (h) Glycerol turnover and (i) glycerol clearance rates before and after metformin treatment. (j) Liver pyruvate carboxylase flux (VPC) and (k) hepatic mitochondrial citrate synthase flux (VCS) in HFD-STZ rats post-metformin treatment versus saline. Data are mean ± SEM, (for a,b,g: saline, n=8, metformin n=9; for e: saline n=12, metformin n=11; for f: saline n=12, metformin n=9; for c,d,h,i: saline n=7, metformin n=7; for j,k: saline n=8, metformin n=8 biological replicates). For statistical analysis, P values were calculated by two-tailed unpaired Student’s t-test (a,b,e-g,j,k) and two-tailed paired Student’s t-test (c,d,h,i), and NS = Not significant.
Figure 6
Figure 6
Chronic oral metformin treatment (3.5 mg ml−1 in the drinking water for 14 days) specifically inhibits contributions from lactate but not alanine to hepatic gluconeogenesis without impacting mitochondrial citrate synthase flux (VCS) in a HFD-STZ Sprague-Dawley rat model of T2D. (a) Fasting plasma glucose concentrations in chronic oral metformin treated rats from both [3-13C]lactate and [3-13C]alanine tracer infusion cohorts. (b) Hepatic cytosolic redox state as determined by the [lac]:[pyr] ratio. (c) Liver G-3-P concentrations in chronically treated metformin or saline rats post tracer infusion. (d) EGP rates in rats from both the [3-13C]lactate and [3-13C]alanine tracer infusion cohorts. (e) Liver pyruvate carboxylase flux (VPC) and (f) hepatic mitochondrial citrate synthase flux (VCS). (g) Contribution of lactate to glucose indicated by the metformin/saline ratio of glucose labeled in the 1, 2, 5 and 6 positions from [3-13C] lactate. Contribution of alanine is indicated by the metformin/saline ratio of labeling in the 1, 2, 5 and 6 positions of glucose from [3-13C] alanine. Data are mean ± SEM (a–f) or mean ± SD (g). (For a,d,g: [3-13C]lactate, saline: n=7, metformin: n=6; [3-13C]alanine, saline: n=6, metformin: n=9,; for b,c: saline: n=13, metformin: n=15; for e,f: saline: n=9, metformin: n=8 biological replicates). For statistical analysis, P values were calculated by two-way ANOVA (a,d), unpaired two-tailed t-test with Welch’s correction (ac,e,f) and effect size d by Cohen’s standard (g) and NS = not significant.

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References

    1. Owen MR, DE, Halestrap AP. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J. 2000;348(Pt 3):607–614. - PMC - PubMed
    1. El-Mir M-Y, NV, Fontaine E, Averet N, Rigoulet M, Leverve X. Dimethylbiguanide Inhibits Cell Respiration via an Indirect Effect Targeted on the Respiratory Chain Complex I. J Biol Chem. 2000;275:223–228. - PubMed
    1. He L, SA, Djedjos S, Miller R, Sun X, Hussain MA, Radovick S, Wondisford FE. Metformin and insulin suppress hepatic gluconeogenesis through phosphorylation of CREB binding protein. Cell. 2009;137:635–646. - PMC - PubMed
    1. Cao J, MS, Chang E, Beckwith-Fickas K, Xiong L, Cole RN, Radovick S, Wondisford FE, He L. Low concentrations of metformin suppress glucose production in hepatocytes through AMP-activated protein kinase (AMPK) J Biol Chem. 2014;289:20435–20446. - PMC - PubMed
    1. Fullerton MD, GS, Marcinko K, Sikkema S, Pulinilkunnil T, Chen ZP, O’Neill HM, Ford RJ, Palanivel R, O’Brien M, Hardie DG, Macaulay SL, Schertzer JD, Dyck JR, van Denderen BJ, Kemp BE, Steinberg GR. Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulin-sensitizing effects of metformin. Nat Med. 2013;19:1649–1654. - PMC - PubMed

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