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, 289 (30), 20435-46

Low Concentrations of Metformin Suppress Glucose Production in Hepatocytes Through AMP-activated Protein Kinase (AMPK)

Low Concentrations of Metformin Suppress Glucose Production in Hepatocytes Through AMP-activated Protein Kinase (AMPK)

Jia Cao et al. J Biol Chem.

Abstract

Metformin is a first-line antidiabetic agent taken by 150 million people across the world every year, yet its mechanism remains only partially understood and controversial. It was proposed that suppression of glucose production in hepatocytes by metformin is AMPK-independent; however, unachievably high concentrations of metformin were employed in these studies. In the current study, we find that metformin, via an AMP-activated protein kinase (AMPK)-dependent mechanism, suppresses glucose production and gluconeogenic gene expression in primary hepatocytes at concentrations found in the portal vein of animals (60-80 μM). Metformin also inhibits gluconeogenic gene expression in the liver of mice administered orally with metformin. Furthermore, the cAMP-PKA pathway negatively regulates AMPK activity through phosphorylation at Ser-485/497 on the α subunit, which in turn reduces net phosphorylation at Thr-172. Because diabetic patients often have hyperglucagonemia, AMPKα phosphorylation at Ser-485/497 is a therapeutic target to improve metformin efficacy.

Figures

FIGURE 1.
FIGURE 1.
Metformin inhibited gluconeogenic gene expression in mouse primary hepatocytes. a–d, pretreatment (Pre-T) with the indicated amounts of metformin (Met) for 3 or 24 h prior to the addition of 0.2 mm Bt-cAMP had a much stronger effect on glucose production (a), G6pc (b), Pck1 (c), and Fbp1 (d) mRNA levels than the treatment in which metformin and Bt-cAMP were added simultaneously. Primary hepatocytes were treated as described under “Experimental Procedures” (n = 4). Same-T, same-time treatment. e, pretreatment of metformin for 3 h greatly activated AMPK (p-AMPKα) and had a stronger inhibition of 0.2 mm Bt-cAMP (4-h incubation)-stimulated G6pc and Pck1 protein levels in primary hepatocytes. * signifies that groups with the same treatment are significantly different (p < 0.05). Error bars indicate mean ± S.D.
FIGURE 2.
FIGURE 2.
Metformin suppressed gluconeogenic gene expression in the liver of DIO mice. a–c, administration of metformin (50 mg/kg of body weight) for 6 weeks significantly decreased mRNA levels of G6pc (a) and Pck1 (b) in the liver of DIO mice (n = 4). c, the mRNA levels of GAPDH in the liver of same DIO mice as in a and c. Error bars indicate mean ± S.D. RD, regular diet; HFD, high fat diet.
FIGURE 3.
FIGURE 3.
Depletion of AMPK catalytic α subunits decreased the effect of metformin to inhibit glucose production in primary hepatocytes. a, two sets of LKB1 adenoviral (ad-shLKB1#1 and ad-shLKB1#2) shRNAs were added in Hepa1–6 cells for 48 h followed by the addition of 0.25 mm metformin (Met) for 6 h. ad-shSCR, adenoviral scramble shRNA; p-AMPK, phospho-AMPK. b, 48 h after the addition of adenoviral shRNAs to deplete AMPKα1 and AMPKα2, primary hepatocytes were treated with the indicated amount of metformin for 3 h during serum starvation and then incubated with 0.2 mm Bt-cAMP and metformin for 4 h. ACC, acetyl-CoA carboxylase; pACC, phospho-ACC; pCREB, phospho-CREB. c, 48 h after adenoviral shRNAs mediated depletion of AMPKα1 and AMPKα2, metformin was added to medium during serum starvation. After washing with PBS, both metformin and 0.2 mm Bt-cAMP were added in glucose production medium (n = 3). Pre-T, pretreatment. * signifies that groups with the same treatment are significantly different (p < 0.05). Error bars indicate mean ± S.D.
FIGURE 4.
FIGURE 4.
A decrease in cAMP levels is not necessary for suppression of glucose production by metformin. a, the AMP/ATP ratio in primary hepatocytes treated with Bt-cAMP and the indicated amount of metformin (Met) simultaneously for 3 h (n = 4). b and c, primary hepatocytes were treated with Bt-cAMP and metformin simultaneously for 3 h. A high concentration of metformin (1 mm) decreased ATP (b) and total nucleotide levels (c) (n = 4). d, metformin did not decrease the cAMP levels in primary hepatocytes pretreated with metformin for 3 h and then treated with metformin and 0.2 mm Bt-cAMP together for another 3 h (n = 4). e and f, metformin significantly suppressed glucagon-stimulated glucose production at 0.25, 0.5, and 1 mm (e) after 4 h of 5 nm glucagon together with the indicated metformin treatment; however, only a high concentration of metformin (1 mm) significantly decreased cAMP levels (f) in primary hepatocytes (n = 4). Error bars indicate mean ± S.D.
FIGURE 5.
FIGURE 5.
Activation of AMPK by low metformin concentrations inhibited gluconeogenic gene expression. a, pretreatment with low concentration of metformin (Met, 80 μm) increased the phosphorylation of AMPKα (p-AMPK) at Thr-172. Hepa1–6 cells were grown in DMEM medium (5.5 mm glucose) plus 10% FBS and metformin for the indicated times. b, AMPK activity in primary hepatocytes after 24 h of metformin treatment (n = 3). c and d, primary hepatocytes were grown in DMEM medium plus 10% FBS and metformin for 21 h and then FBS-free DMEM and metformin for 3 h followed by glucose production medium supplemented with 0.2 mm Bt-cAMP and metformin for another 3 h. Glucose production was measured in the medium (c) (n = 4), and cellular lysates were subjected to immunoblot (d). pCREB, phospho-CREB. e–g, suppression of the Bt-cAMP-stimulated mRNA levels of G6pc (e), Pck1 (f), and GAPDH (g) in the primary hepatocytes pretreated with 80 μm metformin and then with Bt-cAMP (n = 4). h, in primary hepatocytes, 36 h after the addition of adenoviral shRNAs of AMPKα1 and AMPKα2 (ad-shAMPK1/2), metformin was added into DMEM medium plus 10% FBS for 21 h and then added to FBS-free DMEM for 3 h and glucose production medium together with 0.2 mm Bt-cAMP for another 3 h (n = 3). * signifies that groups with the same treatment are significantly different (p < 0.05). NS, not significant. Error bars indicate mean ± S.D. ad-shSCR, adenoviral scramble shRNA.
FIGURE 6.
FIGURE 6.
Low metformin concentration increased ATP levels in primary hepatocytes. a–c, metformin (Met) was added into DMEM medium plus 10% FBS for 21 h and then added to FBS-free DMEM for 3 h and glucose production medium together with 0.2 mm Bt-cAMP for another 3 h. Metformin increased the total nucleotides (a) and ATP levels (b) without affecting AMP/ATP ratio (c) and adenylate energy charge (d) (n = 6). * signifies that groups with the same treatment are significantly different (p < 0.05). Error bars indicate mean ± S.D.
FIGURE 7.
FIGURE 7.
Suppression of glucagon-stimulated glucose production by low metformin concentration in primary hepatocytes. a and b, pretreatment with 80 μm metformin (Met) for 24 h significantly inhibited 5 nm glucagon-stimulated glucose production in primary hepatocytes (a) without affecting the cAMP levels (b). c–e, total nucleotides (c), ATP levels (d), AMP/ATP ratio (e), and adenylate energy charge (f) in primary hepatocytes treated as in a and b (n = 3). g, in primary hepatocytes, 36 h after the addition of adenoviral shRNAs of AMPKα1 and AMPKα2, metformin was added into DMEM medium plus 10% FBS for 21 h and then added to FBS-free DMEM for 3 h and glucose production medium together with 5 nm glucagon for another 3 h (n = 3). * signifies that groups with the same treatment are significantly different (p < 0.05). NS, not significant. Error bars indicate mean ± S.D.
FIGURE 8.
FIGURE 8.
The cAMP-PKA pathway negatively regulates AMPK activity. a, primary hepatocytes were treated with 0.25 mm metformin 3 h prior to the addition of 0.2 mm Bt-cAMP or metformin and Bt-cAMP at the same time (n = 3). Same-T, same treatment; Pre-T, pretreatment. b, in primary hepatocytes, 0.25 mm metformin (Met) was added to FBS-free medium during serum starvation in pretreatment group (Pre-Met). After washing with PBS, both 0.25 mm metformin and 0.2 mm Bt-cAMP were added in glucose production medium for another 3 h. p-AMPKα, phospho-AMPKα; ACC, acetyl-CoA carboxylase; pACC, phospho-ACC; pCREB, phospho-CREB. c, primary hepatocytes were treated with 0.5 mm metformin or together with 0.2 mm Bt-cAMP and harvested at the indicated time points. d, Hepa1–6 cells were grown in DMEM medium (5.5 mm glucose) plus 10% FBS for 16 h and then subjected to serum starvation for 2 h, and 0.5 mm metformin and the indicated amounts of glucagon were added for 4 h. e, 48 h after the addition of adenoviral FLAG-tagged AMPKα1, Hepa1–6 cells were incubated with 0.5 mm metformin and 0.2 mm Bt-cAMP for 4 h. ip, immunoprecipitation. f, blockade of PKA by H89 increased phosphorylation of AMPKα at Thr-172. Primary hepatocytes were treated with 20 μm H89 or vehicle (Veh) for 30 min prior to the addition of 100 nm glucagon. Cells were incubated for 3 h before harvest. g, the PKA competitive inhibitor-(Rp)-cAMP (100 μm) also blocked the negative effect of Bt-cAMP on AMPK phosphorylation at Thr-172. (Rp)-cAMP was administrated 1 h before the addition of 0.2 mm Bt-cAMP in Hepa1–6 cells. h, H89 accentuated calyculin (protein phosphatase inhibitor)-mediated phosphorylation of AMPKα1 at Thr-172. Primary hepatocytes were incubated with H89 or H89 plus 50 nm calyculin (Cell Signaling) for 75 min before harvest. Error bars indicate mean ± S.D.
FIGURE 9.
FIGURE 9.
Phosphorylation of AMPKα at Ser-485/497 by cAMP decreased metformin-mediated AMPK activation. a, FLAG-tagged AMPKα1 was immunoprecipitated (FLAG ip) from primary hepatocytes treated with 0.2 mm Bt-cAMP and was used to map the phosphorylation site(s). p-AMPKα, phospho-AMPKα; pCREB, phospho-CREB. b and c, identification of AMPKα1 phosphorylation at Ser-485/497 by cAMP in LCMS/MS analysis. d, purified AMPKα1 was incubated with different amounts of PKA catalytic subunit (PKAc; Millipore) at 30 °C for 30 min. e and f, 48 h after the addition of adenoviral FLAG-tagged AMPKα1 and its mutants, Hepa1–6 cells were subjected to 90 min of serum starvation, and then 0.5 mm metformin (Met) and 0.2 mm Bt-cAMP were added for 4 h. FLAG-tagged AMPKα1 and its mutants were expressed at levels 5-fold higher than endogenous α1 levels.
FIGURE 10.
FIGURE 10.
AMPKα1 mutant (S485A) abolished the negative effect of cAMP on AMPKα1 activation. a, 48 h after the addition of adenoviral FLAG-tagged AMPKS485A mutant in Hepa1–6 cells, 0.5 mm metformin (Met) and 0.2 mm Bt-cAMP were added, and cells were harvested 5 h after the incubation. FLAG-tagged AMPKS485A was purified by using FLAG immunoprecipitation (FLAG ip) kit (Sigma). pCREB, phospho-CREB. b, 36 h after adenoviral FLAG-tagged AMPKα1 and S485A mutant were added to primary hepatocytes, cells underwent 3 h of serum starvation, and then medium was changed to glucose production medium supplemented with 0.25 mm metformin and 0.2 mm Bt-cAMP for another 4 h (n = 3). p-AMPKα1, phospho-AMPKα1. * signifies that groups with the same treatment are significantly different (p < 0.05). NS, not significant. Error bars indicate mean ± S.D.

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