Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase

Abstract

Metformin is considered to be one of the most effective therapeutics for treating type 2 diabetes because it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain or posing a risk of hypoglycaemia. For over half a century, this agent has been prescribed to patients with type 2 diabetes worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production, while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide knockdown of hepatic mitochondrial glycerophosphate dehydrogenase in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decreases in plasma glucose concentrations, and inhibition of endogenous glucose production. These findings were replicated in whole-body mitochondrial glycerophosphate dehydrogenase knockout mice. These results have significant implications for understanding the mechanism of metformin's blood glucose lowering effects and provide a new therapeutic target for type 2 diabetes.

Figure 1

Effects of acute metformin (50 mg/kg, IV) treatment in Sprague-Dawley rats on plasma glucose, EGP and hepatic redox state. (a) Metformin acutely reduced fasting plasma glucose and (b) EGP. These changes were associated with (c) increased plasma [lactate]:[pyruvate] and (d) decreased plasma [β-hydroxybutyrate]:[acetoacetate]. (e) Increased hepatic cytosolic redox state, (f) decreased liver mitochondrial redox state and (g) a tendency to increased plasma glycerol were observed. Data set shown is representative of 3 experiments. Data are mean ± SEM, ((a) saline n=3, metformin n=4; (b–d, g) n=4; (e,f) n=6; biological replicates). * P<0.05, **P<0.01, ***P<0.001 by unpaired t-test.

Figure 2

Effects of chronic metformin (50 mg/kg, IP per day x 30 days) treatment in Sprague-Dawley rats on plasma glucose, EGP and hepatic redox state. (a) Chronic metformin treatment reduced fasting plasma glucose and (b) EGP. These changes were associated with (c) increased plasma [lactate]:[pyruvate], and (d) decreased plasma [β-hydroxybutyrate]:[acetoacetate]. Chronic metformin treatment (e) elevated hepatic cytosolic redox state, (f) decreased liver mitochondrial redox state and (g) increased plasma glycerol. Data are mean ± SEM, ((a) n=5; (b, d–g) saline n=6, metformin n=4; (c) n=4; biological replicates). * P<0.05, **P<0.01, ***P<0.001 by unpaired t-test.

Figure 3

Guanides/biguanides non-competitively inhibit rat and human mGPD activity. (a) Guanides/biguanides inhibit rat mGPD activity by 30–50% in rat mitochondrial lysate. Data shown is the average of 3 experiments. (b) Metformin (50 μM) inhibited mitochondrial respiration from glycerol-3-phosphate. (c) Metformin inhibited recombinant human mGPD non-competitively as determined by Michaelis-Menten and Eadie-Hofstee plots of the kinetic data. Data shown is representative of 2 experiments. Data are mean ± SEM, (n=5 technical replicates for mGPD activity in lysate; nontreat n=12, metformin n=12 technical replicates for oxygen consumption). * P<0.05, **P<0.01, ***P<0.001 by unpaired t-test.

Figure 4

Effects of mGPD and cGPD ASO treatment in Sprague-Dawley rats on metformin action. (a) Knockdown of hepatic mGPD, but not cGPD, by two ASOs reduced fasting plasma glucose. (b) mGPD ASO treatment increased liver [lactate]:[pyruvate], and (c) decreased liver [β-hydroxybutyrate]:[acetoacetate]. cGPD ASO treatment had a less marked effect on both liver redox ratios. (d) mGPD knockdown decreased EGP and abrogated metformin’s effect on EGP. (e) Knockdown of hepatic mGPD decreased plasma glucose (f) increased plasma [lactate]:[pyruvate], (g) decreased plasma [β-hydroxybutyrate]:[acetoacetate] and abrogated metformin’s effect on these parameters. (h) In mGPD ASO treated rats, chronic metformin treatment had no effect on plasma glucose, (i) or EGP. mGPD knockout mice had (j) decreased plasma glucose, (k) reduced EGP, and did not respond to acute metformin treatment. Data are mean ± SEM, ((a) n=6; (b) Control n=6, mGPD n=5, cGPD n=5; (c) Control n=6, mGPD n=5, cGPD n=6; (d) n=5; (e) Control n=4, mGPD n=6; (f,g) Control n=4, mGPD n=5; (h,i) n=7; (j) WT n=13, KO n=4; (k) WT pre-metformin n=9, WT metformin n=13, KO n=4; biological replicates). * P<0.05, **P<0.01, ***P<0.001 by ANOVA; # P<0.05, ##P<0.01, ###P<0.001 by ANOVA.

Extended Data Figure 1

Effect of acute galegine treatment and acute AMPK activator treatment in vivo. A 20-minute infusion of galegine in rats (a) decreased fasting plasma glucose concentrations and (b) decreased fasting plasma insulin concentrations (c) increased plasma lactate concentrations, (d) independently of any changes in gluconeogenic gene expression. (e) AMPK was activated by acute galegine treatment. (f) 20-minute infusions of A-769662 in rats activated had no effect on fasting plasma glucose concentrations (g) or endogenous glucose production, in spite of (h) comparable activation of AMPK. Data are mean ± SEM, (saline, n=6; galegine, n=8; A-769662, n=5 biological replicates). * P<0.05, **P<0.01, ***P<0.001 by unpaired t-test.

Extended Data Figure 2

Effect of guanide/biguanide treatment on enzymes involved in pyruvate metabolism, redox regulation, and the malate-aspartate shuttle and on complex I mediated respiration. (a) Lactate enters metabolism through LDH via a redox-dependent reaction into the pyruvate pool. Pyruvate lies at the intersection of alanine influx, glycolysis, citric acid cycle and gluconeogenic flux. (b) Guanides/biguanides did not affect pyruvate carboxylase (PC) activity, compared to the known inhibitor coenzyme A, (c) and did not affect citrate synthase activity. (d) ALAT activity was also unaffected. (e) Guanides/biguanides did not affect MDH activity, (f) ASAT activity or (g) total shuttle rates. (h) Metformin had no effect on complex I mediated respiration in isolated mitochondria at concentrations less than 5 mM, and induced a slight increase in complex II respiration. Data are mean ± SEM, (n=5 technical replicates). * P<0.05, **P<0.01, ***P<0.001 by unpaired t-test.

Extended Data Figure 3

Effect of acute metformin (20 mg/kg and 50 mg/kg, IV) treatment on EGP, liver redox, energy charge and liver gluconeogenic protein expression in Sprague-Dawley rats. (a) Acute metformin (20 mg/kg) treatment significantly lowered EGP, (b) increased the liver [lactate]:[:pyruvate] ratio and (c) decreased the liver [β-hydroxybutyrate]:[acetoacetate]. (d) Acute metformin (50 mg/kg) treatment increased liver [GSSG]:[GSH], (e) but had no effect on the liver [ATP]:[ADP] or (f) [ATP]:[AMP] ratios. (g) The [NADH]:[NAD⁺] and (h) [NADPH]:[NADP⁺] ratios also remained unchanged. (i) Acute metformin treatment had no effect on liver [cAMP] levels. (j) Acute metformin (50 mg/kg) treatment had no effect on the expression of key gluconeogenic enzymes or AMPK in the liver. (k) PEPCK-C protein expression or (l) PC protein expression were unchanged, though (m) activated CREB as determined by the ratio of phosphorylated CREB to total CREB levels was slightly increased. (n) There was no activation of liver AMPK as reflected by the ratio of phosphorylated AMPK to total AMPK levels, (o) and no change in the phosphorylation of AMPK downstream target ACC. Data are mean ± SEM, (n=6 biological replicates). * P<0.05, **P<0.01, ***P<0.001 by unpaired t-test.

Extended Data Figure 4

Effect of chronic metformin (50 mg/kg per day, IP x 30 days) treatment on liver redox, energy charge and expression of gluconeogenic regulators. (a) Chronic metformin treatment increased the liver [GSSG]:[GSH] ratio, (b) but had no effect on the liver [ATP]:[ADP] or (c) [ATP]:[AMP] ratios. (d) The [NADH]:[NAD⁺] and (e) [NADPH]:[NADP⁺] ratios also remained unchanged. (f) Chronic metformin treatment slightly reduced liver [cAMP] levels. (g) Chronic metformin treatment had no effect on the protein levels of major gluconeogenic enzymes in the liver, (h) PEPCK-C protein expression and (i) liver PC protein levels both remaining unaltered. (j) Activated CREB as determined by the ratio of phosphorylated CREB to total CREB levels was decreased. (k) Chronic metformin treatment activated liver AMPK as indicated by the increased ratio of phosphorylated AMPK to total AMPK levels and (l) increased phosphorylation of ACC. Data are mean ± SEM, (n=6 biological replicates). * P<0.05, **P<0.01, ***P<0.001 by unpaired t-test.

Extended Data Figure 5

Effect of metformin on the glycerophosphate shuttle. NADH made in the cytosol via glycolysis cannot cross the mitochondrial membrane and contribute electrons to the electron transport chain (ETC) for ATP synthesis. Two mechanisms, the reversible malate-aspartate shuttle, and (a) the unidirectional glycerophosphate shuttle, oxidize NADH in the cytosol and transport electrons into the mitochondria via metabolic intermediates. The glycerophosphate shuttle is composed of cytosolic and mitochondrial glycerophosphate dehydrogenases, two structurally distinct enzymes. (b) Metformin had no effect on cGPD, which consists of two subunits and catalyzes the conversion of dihydroyacetone phosphate (DHAP) to glycerol-3-phosphate (G-3-P), oxidizing one NADH. (c) Metformin inhibited the activity of rat mGPD, a FAD⁺-linked enzyme that transmits electron pairs to the ETC via the quinone pool, purified from liver by immunopreciptation. Inhibition of rat mGPD was non-competitive. Data shown are the average of 5 separate experiments. (d) Metformin inhibited pure, recombinant human mGPD non-competitively, decreased V_max without affecting K_m. Data shown are representative of 2 experiments. (e) Metformin also inhibited the activity of the bacterial mGPD isoform, Pediococcus sp. α-glycerophosphate oxidase, showing non-competitive kinetics. Data are mean ± SEM, (n=4–5 technical replicates). * P<0.05, **P<0.01, ***P<0.001 by unpaired t-test.

Extended Data Figure 6

Effect of metformin and knockdown of mGPD by siRNA on glucose production from various substrates in primary hepatocytes, and metformin-mediated increase in glycerol-3-phosphate concentrations [G-3-P]. (a) Metformin treatment (100 μM) and siRNA knockdown of mGPD in rat primary hepatocytes inhibited glucose production at higher [lactate]:[pyruvate] ratios, but lower at redox state induced by decreased [lactate]:[pyruvate] abrogated the ability of metformin and mGPD knockdown by siRNA to decrease glucose production. Decreasing redox state itself inhibited glucose production. (b) Metformin only inhibited glucose production from lactate and glycerol, but not from substrates that do not increase cytosolic redox state. (c) mGPD knockdown by siRNA showed a similar substrate-selective inhibition of glucose production. (d) Both metformin and mGPD siRNA treatment increased [G-3-P] levels in hepatocytes, and (e) acute metformin (50 mg/kg, IV) treatment in vivo increased liver [G-3-P] levels without significantly altering [G-3-P] levels in other tissues, suggesting an impasse at the mGPD catalytic step. (f) siRNA treatment did not induce cytotoxicity as determined by trypan blue exclusion, (g) CyQuant proliferation assay and (h) the absence of cytochrome c release into the cytosolic fraction from mitochondria of treated cells. Data are mean ± SEM, (n=5 technical replicates, n=3 for cytotoxicity tests (f–h)). * P<0.05, **P<0.01, ***P<0.001 by unpaired t-test.

Extended Data Figure 7

Effect of mGPD and cGPD ASO treatment on liver redox, high-energy intermediates and expression of gluconeogenic regulators. (a) mGPD ASO effectively reduced expression of liver mGPD protein and (b) cGPD ASO effectively reduced liver cGPD protein levels. (c) mGPD ASO treatment increased plasma lactate concentrations significantly, but cGPD ASO knockdown had no effect on plasma lactate concentrations. (d) mGPD ASO knockdown increased the liver [GSSG]:[GSH] ratio, (e) had no effect on the liver [ATP]:[ADP], (f) [ATP]:[AMP], (g) [NADH]:[NAD⁺] or (h) [NADPH]:[NADP⁺] ratios, (i) though liver [cAMP] levels were slightly decreased. (j) ASO-mediated knockdown of mGPD did not affect expression of gluconeogenic enzymes, (k) PEPCK-C and (l) PC protein levels remaining unchanged in the liver. (m) Activated CREB as determined by the phosphorylated CREB was decreased, (n) and mGPD ASO knockdown led to activation of liver AMPK as indicated by increased phosphorylated AMPK and (o) increased ACC phosphorylation. Data are mean ± SEM, (n=6 biological replicates). * P<0.05, **P<0.01, ***P<0.001 by unpaired t-test.

Extended Data Figure 8

Computational binding model of guanides/biguanides to mGPD from Streptococcus sp. Modeling of guanides/bigaunides binding to mGPD after modification of key residues to fit the human sequence, show (a) FAD⁺ binding and predicted movement in the pocket, (b) metformin binding, and (c) phenformin binding to the FAD⁺-containing pocket.

Extended Data Figure 9

Plasma and tissue metformin concentrations in rats treated with metformin. (a) Acute metformin (50 mg/kg, IV) administration led to peak plasma metformin concentrations of approximately 74 μM; 100 mg/kg and 250 mg/kg doses increased plasma metformin concentration to 345 μM and 1300 μM, respectively. (b) Acute metformin (50 mg/kg, IV) led to liver metformin concentrations of approximately 100 μM, and metformin levels in other tissues were comparatively low. Data shown is representative of 2 experiments. Data are mean ± SEM, (n=3 biological replicates for plasma concentrations, n=5 biological replicates for tissue levels). * P<0.05, **P<0.01, ***P<0.001 by ANOVA.

Extended Data Figure 10

Effect of mGPD and cGPD antisense oligonucleotide (ASO) treatment on liver toxicity and tissue-specific knockdown of mGPD expression by mGPD ASO. (a) mGPD ASO treatment had no effect on body weight after treatment at 37.5 mg/kg ASO twice a week for 4 weeks. (b) All ASOs screened in this study, mGPD ASO #1, mGPD ASO #2, cGPD ASO #1 and cGPD ASO #2 elicited no significant liver toxicity as determined by plasma AST/ALT levels after 4 weeks. (c) Treatment with mGPD ASO #2 for 4 weeks during the mGPD ASO with acute metformin study also had no effect on plasma AST/ALT. (d) mGPD ASO treatment led to cleavage of mGPD mRNA transcript exclusively in the liver, only slightly decreasing transcript levels in white adipose tissue and having no effect on mGPD mRNA in other tissues. (e–k) mGPD ASO treatment specifically reduced protein expression of mGPD in the liver, with no significant effect on mGPD protein levels in the pancreas, kidney, muscle, white adipose or brown adipose. Data are mean ± SEM, (n=6 biological replicates). * P<0.05, **P<0.01, ***P<0.001 by unpaired t-test.