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, 24 (9), 1395-1406

Metformin Reduces Liver Glucose Production by Inhibition of fructose-1-6-bisphosphatase

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Metformin Reduces Liver Glucose Production by Inhibition of fructose-1-6-bisphosphatase

Roger W Hunter et al. Nat Med.

Abstract

Metformin is a first-line drug for the treatment of individuals with type 2 diabetes, yet its precise mechanism of action remains unclear. Metformin exerts its antihyperglycemic action primarily through lowering hepatic glucose production (HGP). This suppression is thought to be mediated through inhibition of mitochondrial respiratory complex I, and thus elevation of 5'-adenosine monophosphate (AMP) levels and the activation of AMP-activated protein kinase (AMPK), though this proposition has been challenged given results in mice lacking hepatic AMPK. Here we report that the AMP-inhibited enzyme fructose-1,6-bisphosphatase-1 (FBP1), a rate-controlling enzyme in gluconeogenesis, functions as a major contributor to the therapeutic action of metformin. We identified a point mutation in FBP1 that renders it insensitive to AMP while sparing regulation by fructose-2,6-bisphosphate (F-2,6-P2), and knock-in (KI) of this mutant in mice significantly reduces their response to metformin treatment. We observe this during a metformin tolerance test and in a metformin-euglycemic clamp that we have developed. The antihyperglycemic effect of metformin in high-fat diet-fed diabetic FBP1-KI mice was also significantly blunted compared to wild-type controls. Collectively, we show a new mechanism of action for metformin and provide further evidence that molecular targeting of FBP1 can have antihyperglycemic effects.

Conflict of interest statement

Competing Financial Interests Statements

K.S. is a full-time employee of the Nestlé Institute of Health Sciences S.A., Switzerland.

Figures

Figure 1
Figure 1. Generation of an AMP-insensitive FBP1 knockin mouse model.
(a) Human FBP1 structure (PDBID 1FTA) represented as ribbons and AMP and interacting residues (numbered from the initiator methionine) are shown as sticks. Dashed lines represent hydrogen bonding interactions, whereas residues making hydrophobic contracts are illustrated as sticks and transparent surfaces. Red and blue spheres represent backbone oxygen and nitrogen atoms respectively. (b) Coomassie-stained SDS-PAGE of mouse liver FBPase and recombinant mouse 6HIS-FBP1 preparations with single point mutations designed to disrupt AMP binding. (c) AMP inhibition curves of mouse 6HIS-FBP1 mutants. FBPase activity is expressed as a ratio of the maximum activity in the absence of AMP (V/Vo). IC50 values represent the mean ± SD of three independent measurements on two enzyme preparations. The line graph is representative of the results from a single preparation. (d) Schematic illustrating the targeting strategy used to generate C57BL/6NTac FBP1G27P knockin (KI) mice. Exons and FRT recombination sites are represented by dark grey boxes and triangles respectively. The KI allele containing the G27P mutation in exon 1 is shaded pale grey. Correct recombination was confirmed by Southern blotting of PsiI and KpnI digests of genomic DNA isolated from targeted embryonic stem cells with the corresponding 5’ and 3’ probes (black boxes). Genotyping of the constitutive KI allele was performed by PCR of genomic DNA using primers P1 and P2. (e) Liver biopsies from overnight fasted (16 h) or refed (4 h) FBP1WT/WT (WT) or FBP1G27P/G27P (KI) mice were assayed for Fbp1 mRNA expression by qPCR (left chart) or FBPase activity (right chart) by spectrophotometric assay. The line graph (below) represents AMP inhibition curves of FBPase activity in liver homogenates expressed as a ratio of the activity in the absence of AMP (V/Vo). Results represent mean ± SD, n = 5-7 per group.
Figure 2
Figure 2. FBP1 G27P knockin mice display normal glucose homeostasis.
(a-e) FBP1WT/WT (WT) or FBP1G27P/G27P (KI) mice were fasted overnight for 16 h (Fasted) or subsequently given free access to standard chow for 4 h (Refed). Blood and liver biopsies were taken and the following parameters determined: blood glucose (a), plasma insulin (b), plasma glucagon (c), plasma leptin (d) and liver glycogen (e). n = 5-7 (WT) and 5-7 (KI) per group. (f) Glucose (2 g.kg-1 p.o.) and (g) pyruvate (1 g.kg-1 i.p.) tolerance was assessed on mice fasted for 16 h. Results represent mean ± SE, n = 10 per group. (h) Expression of the major enzymes and regulatory components of the gluconeogenic, glycogenic and glycogenolytic pathways in liver samples from fasted or refed animals was determined by Western blotting. Representative results from three mice per group are shown. *P < 0.05 (Fasted vs. refed). Statistical significance was determined using unpaired, two-tailed Student’s t-test and an alpha level of 0.05.
Figure 3
Figure 3. FBP1 G27P KI mice are resistant to the hypoglycemic action of an AMP-mimetic FBPase inhibitor.
(a) Diagram showing the structure of MB06322 and the active metabolite, MB05032. (b) Mouse and rat FBP1 preparations were assayed for inhibition by AMP (closed symbols) and MB05032 (open symbols). Results represent mean ± SD, n = 3. *P < 0.05 (Mouse vs. rat) (c-g) Vehicle (10:10:80 Solutol HS 15:PEG 400:water) or MB06322 (75 mg.kg-1 i.p.) was administered to fasted (16 h) WT (c) or KI (f) mice and blood glucose monitored at the indicated intervals for 3 h. (d, g) Lactate was measured just prior to drug administration and at t = 120 min using a lactate meter. (e) Plasma levels of MB05032 were assayed from blood samples drawn at the end of the protocol (t = 180 min). Results represent mean ± SE, n = 4-5 per treatment group. *P < 0.05 (Vehicle vs. MB06322). Statistical significance was determined using unpaired, two-tailed Student’s t-test and an alpha level of 0.05.
Figure 4
Figure 4. FBP1 G27P KI mice are resistant to the hypoglycemic action of AICAR.
(a) Diagram illustrating the structure of AMP and ZMP. (b-l) AICAR tolerance was determined by administering vehicle (0.9 % saline) or AICAR (250 mg.kg-1- i.p.) to fasted (16 h) WT (b, e, g, i) or KI (c, f, h, j) mice. Blood glucose was measured at the indicated timepoints for a period of 3 h. (d) Plasma AICAR, (e, f) blood lactate, (g, h) plasma glucagon and (i, j) plasma insulin were assayed from blood samples drawn at t = 60 min. Results represent mean ± SE, n = 4-8 per treatment group. *P < 0.05 (Vehicle vs. AICAR). #P < 0.05 (WT vs. KI). (k, l) Vehicle (0.9 % saline) or AICAR (250 mg.kg-1- i.p.) was administered to fasted (16 h) mice and after 60 min exposure, liver biopsies were taken and assayed for AMPK activation by Western blotting. The blot image depicts three representative mice from each treatment group and a quantitative analysis of pT172 AMPKα phosphorylation from the entire sample set is shown in (l). Results are expressed as pT172 AMPKα/AMPKα ratio normalized to the WT-vehicle group. n = 4-5 per treatment group. *P < 0.05 (Vehicle vs. AICAR). Statistical significance was determined using unpaired, two-tailed Student’s t-test and an alpha level of 0.05.
Figure 5
Figure 5. FBP1 G27P KI mice exhibit resistance to the acute glucose-lowering effect of metformin.
(a, b) Vehicle (water) or metformin (250 mg.kg-1 p.o.) was administered to fasted (16 h) mice and after 45 min, glucose tolerance (2 g.kg-1 i.p.) was assessed by monitoring blood glucose over a period of 2 h. Results represent mean ± SE, n = 18 (WT-vehicle), 17 (WT-metformin), 15 (KI-vehicle) and 15 (KI-metformin). *P < 0.05 (vehicle vs. metformin). (c-e) Mice were fasted for 16 h and dosed with vehicle (water) or metformin (250 mg.kg-1 p.o.). After 1 h exposure, blood and liver biopsies were taken and assayed for metformin (c). (d) Western blotting of ACC and AMPKα phosphorylation in livers from vehicle and metformin-treated mice. Representative results from three mice per group are shown. (e) Quantitative analysis of pT172 AMPKα. Results are expressed as pT172 AMPKα/AMPKα ratio normalized to the WT-vehicle group. n = 5. (f) Arterial blood glucose and glucose infusion rate (GIR) (g) during metformin-euglycemic clamps in FBP1WT/WT (WT) or FBP1G27P/G27P (KI) mice. Animals were fasted for 5 h and infused i.v. with metformin (3.75 mg.kg-1.min-1) and a variable infusion of 50 % glucose to maintain euglycemia at 120 mg.dl-1 over a period of 120 min. *P < 0.05 (WT vs. KI). (h) Plasma and liver metformin concentrations at the end of the clamp period. (i-l) Rates of endogenous glucose production (EndoRa) (i), gluconeogenesis (GNG) (j), glycogenolysis (GYG) (k) and glucose disappearance (Rd) (l) during the resting period (5 h fasted) and steady state of the metformin clamp (average from 100-120 min). Results represent mean ± SE, n = 8 (WT-resting), 8-9 (WT-clamp), 10 (KI-resting) and 9-11 (KI-clamp). *P < 0.05. Statistical significance was determined using unpaired, two-tailed Student’s t-test and an alpha level of 0.05.
Figure 6
Figure 6. FBP1 G27P KI mice are resistant to the glucose lowering effects of metformin in an obesity-induced model of diabetes.
Body mass (a) and cumulative food intake (b) for FBP1WT/WT (WT) and FBP1G27P/G27P (KI) fed a 60 % Kcal high fat diet ad libitum over a period of eight weeks. (c-f) Glucose tolerance (1.5 g.kg-1 i.p.) (Glycemia in c and corresponding AUC in d), plasma insulin (e) and triglyceride (TG) (f) were assessed after eight weeks of dietary intervention. Results represent mean ± SE (a-c), n = 10-12 per group. (g, h) After 10 weeks of dietary intervention mice were fasted for 16 h, administered vehicle (water) or metformin (250 mg.kg-1 p.o.) and blood glucose measured after 2 h. n =13 (WT-vehicle), 15 (WT-metformin), 8 (KI-vehicle) and 12 (KI-metformin). *P < 0.05 (Vehicle vs. metformin). #P < 0.05 (Resting vs. 120 min) (i-k) After 12 weeks of dietary intervention mice were fasted for 16 h, administered vehicle (water) or metformin (250 mg.kg-1 p.o.) and liver biopsies were taken after 2 h of drug treatment. Liver metformin (i), pT172 AMPKα phosphorylation (expressed as pT172 AMPKα/AMPKα ratio normalized to the WT-vehicle group) (j), and cAMP (k) are shown. n = 6-7 (WT-vehicle), 6 (WT-metformin), 6-7 (KI-vehicle) and 6 (KI-metformin). *P < 0.05 (Vehicle vs. metformin). #P < 0.05 (WT vs KI). Statistical significance was determined using unpaired, two-tailed Student’s t-test and an alpha level of 0.05.

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