Metformin improves vascular and metabolic insulin action in insulin-resistant muscle

in Journal of Endocrinology
Correspondence should be addressed to M A Keske: Michelle.Keske@deakin.edu.au

*(E A Bradley and D Premilovac contributed equally to this work)

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Insulin stimulates glucose disposal in skeletal muscle in part by increasing microvascular blood flow, and this effect is blunted during insulin resistance. We aimed to determine whether metformin treatment improves insulin-mediated glucose disposal and vascular insulin responsiveness in skeletal muscle of insulin-resistant rats. Sprague–Dawley rats were fed a normal (ND) or high-fat (HFD) diet for 4 weeks. A separate HFD group was given metformin in drinking water (HFD + MF, 150 mg/kg/day) during the final 2 weeks. After the intervention, overnight-fasted (food and metformin removed) anaesthetised rats underwent a 2-h euglycaemic–hyperinsulinaemic clamp (10 mU/min/kg) or saline infusion. Femoral artery blood flow, hindleg muscle microvascular blood flow, muscle glucose disposal and muscle signalling (Ser473-AKT and Thr172-AMPK phosphorylation) were measured. HFD rats had elevated body weight, epididymal fat pad weight, fasting plasma insulin and free fatty acid levels when compared to ND. HFD-fed animals displayed whole-body and skeletal muscle insulin resistance and blunting of insulin-stimulated femoral artery blood flow, muscle microvascular blood flow and skeletal muscle insulin-stimulated Ser473-AKT phosphorylation. Metformin treatment of HFD rats reduced fasting insulin and free fatty acid concentrations and lowered body weight and adiposity. During euglycaemic-hyperinsulinaemic clamp, metformin-treated animals showed improved vascular responsiveness to insulin, improved insulin-stimulated muscle Ser473-AKT phosphorylation but only partially restored (60%) muscle glucose uptake. This occurred without any detectable levels of metformin in plasma or change in muscle Thr172-AMPK phosphorylation. We conclude that 2-week metformin treatment is effective at improving vascular and metabolic insulin responsiveness in muscle of HFD-induced insulin-resistant rats.

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  • Supplementary Figure 1: Example immunoblot of skeletal muscle total Akt (panel A) and Ser473Akt (panel B). ND = normal diet fed; HFD = high fat diet fed; HFD+MF = HFD+metformin; SAL = saline; INS = insulin.
  • Supplementary Figure 2: Example immunoblot of skeletal muscle total AMPK (panel A) and Thr172AMPK(panel B). EX CTRL = exercised muscle as a positive control; ND = normal diet fed; HFD = high fat diet fed; HFD+MF = HFD+metformin; SAL = saline; INS = insulin.
  • Supplementary Figure 3: Mean arterial pressure time course during a 2 hour euglycemic hyperinsulinemic clamp (10mU/min/kg) or saline infusion in normal diet (ND, panel A), high fat diet (HFD, panel B) and high fat diet + Metformin (HFD + MF, panel C) treated rats. Mean arterial pressure at the conclusion of the 2 hour experiment in ND, HFD and HFD+MF rats (panel D). Data are means ± SEM for n= 14-17 rats in each group. Two-way-ANOVA.
  • Supplementary Figure 4: Heart rate time course during a 2 hour euglycemic hyperinsulinemic clamp (10mU/min/kg) or saline infusion in normal diet (ND, panel A), high fat diet (HFD, panel B) and high fat diet + Metformin (HFD + MF, panel C) treated rats. Heart rate at the conclusion of the 2 hour experiment in ND, HFD and HFD+MF rats (panel D). Data are means ± SEM for n= 14-17 rats in each group. Two-way-ANOVA.

 

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    Experimental protocol. Following surgical preparation and ~60 min of equilibration, a constant infusion of saline or insulin (10 mU/min/kg) was commenced at 0 min and continued for 120 min. Glucose (30% wt./vol.) infusion, initiated shortly after the commencement of insulin infusion, was administered at a rate required to maintain euglycaemia as determined from arterial blood glucose assessment (black teardrops). At 55 min, a bolus injection of allopurinol (10 µmol/kg) was made followed by infusions of 1-methylxanthine (1-MX, 0.4 mg/min/kg) for 60 min. Constant infusions are indicated by the bars. At 75 min a bolus of 2-DG (20 µCi) was administered and radioactive plasma samples were collected at 80, 85, 90, 105 and 120 min to determine the plasma 2-DG clearance. Arterial and femoral vein samples (A-V) were taken 120 min to determine 1-MX metabolism. Calf muscles were freeze clamped and stored at −80°C at the end of the experiment for subsequent determination of muscle glucose uptake (R′g) and AKT and AMPK phosphorylation.

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    Glucose infusion rate (GIR) (panel A) required to maintain fasting blood glucose levels (panel B) during a euglycaemic–hyperinsulinaemic clamp (10 mU/min/kg) for normal diet (ND), high-fat diet (HFD) and high-fat diet + metformin (HFD + MF)-treated rats. Data are means ± s.e.m. for n = 14–17 rats in each group. *P < 0.05 ND vs HFD, # P < 0.05 HFD vs HFD + MF, P < 0.05 ND vs HFD + MF. Two-way repeated-measures ANOVA and SNK post hoc test.

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    Plasma insulin levels before (0 min) and at the end (120 min) of the euglycaemic–hyperinsulinaemic clamp (10 mU/min/kg) for normal diet (ND), high-fat diet (HFD) and high-fat diet + metformin (HFD + MF)-treated rats. Data are means ± s.e.m. for n = 14–17 rats in each group. *P < 0.05 vs 0 min, # P < 0.05 ND at 0 min vs HFD at 0 min. Two-way ANOVA and SNK post hoc test.

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    Skeletal muscle microvascular blood flow (panel A) and muscle specific glucose uptake (R′g) (panel B) at the conclusion of a 2-h euglycaemic–hyperinsulinaemic clamp (10 mU/min/kg) or saline infusion for normal diet (ND), high-fat diet (HFD) and high-fat diet + metformin (HFD + MF) treated rats. Data are means ± s.e.m. for n = 14–17 rats in each group. *P < 0.05 vs saline, # P < 0.05 ND, P < 0.05 HFD. Two-way-ANOVA and SNK post hoc test.

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    Skeletal muscle Western blot analysis of Ser473-AKT phosphorylation (panel A) and Thr172-AMPK (panel B) at the conclusion of a 2-h euglycaemic–hyperinsulinaemic clamp (10 mU/min/kg) or saline infusion for normal diet (ND), high-fat diet (HFD) and high-fat diet + metformin (HFD + MF) treated rats. Data expressed as means ± s.e.m. for n = 7 rats for panel A and n = 3–4 rats in panel B. *P < 0.05 vs saline, # P < 0.05 ND, P < 0.05 HFD. Two-way ANOVA and SNK post hoc test. Dotted line represents the extent of AMPK activation with contracted muscle.

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    Femoral artery blood flow time course during a 2-h euglycaemic–hyperinsulinaemic clamp (10 mU/min/kg) or saline infusion in normal diet (ND, panel A), high-fat diet (HFD, panel B) and high-fat diet + metformin (HFD + MF, panel C)-treated rats. Femoral artery blood flow at the conclusion of the 2 h experiment in ND, HFD and HFD + MF rats (panel D). Data are means ± s.e.m. for n = 14–17 rats in each group. *P < 0.05 vs saline, P < 0.05 HFD. Two-way ANOVA and SNK post hoc test.

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