Nitric oxide increases cyclic GMP levels, AMP-activated protein kinase (AMPK)alpha1-specific activity and glucose transport in human skeletal muscle

Abstract

Aims/hypothesis: We investigated the direct effect of a nitric oxide donor (spermine NONOate) on glucose transport in isolated human skeletal muscle and L6 skeletal muscle cells. We hypothesised that pharmacological treatment of human skeletal muscle with N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) would increase intracellular cyclic GMP (cGMP) levels and promote glucose transport.

Methods: Skeletal muscle strips were prepared from vastus lateralis muscle biopsies obtained from seven healthy men. Muscle strips were incubated in the absence or presence of 5 mmol/l spermine NONOate or 120 nmol/l insulin. The L6 muscle cells were treated with spermine NONOate (20 micromol/l) and incubated in the absence or presence of insulin (120 nmol/l). The direct effect of spermine NONOate and insulin on glucose transport, cGMP levels and signal transduction was determined.

Results: In human skeletal muscle, spermine NONOate increased glucose transport 2.4-fold (p < 0.05), concomitant with increased cGMP levels (80-fold, p < 0.001). Phosphorylation of components of the canonical insulin signalling cascade was unaltered by spermine NONOate exposure, implicating an insulin-independent signalling mechanism. Consistent with this, spermine NONOate increased AMP-activated protein kinase (AMPK)-alpha1-associated activity (1.7-fold, p < 0.05). In L6 muscle cells, spermine NONOate increased glucose uptake (p < 0.01) and glycogen synthesis (p < 0.001), an effect that was in addition to that of insulin. Spermine NONOate also elicited a concomitant increase in AMPK and acetyl-CoA carboxylase phosphorylation. In the presence of the guanylate cyclase inhibitor LY-83583 (10 micromol/l), spermine NONOate had no effect on glycogen synthesis and AMPK-alpha1 phosphorylation.

Conclusions/interpretation: Pharmacological treatment of skeletal muscle with spermine NONOate increases glucose transport via insulin-independent signalling pathways involving increased intracellular cGMP levels and AMPK-alpha1-associated activity.

Fig. 1

Effect of spermine nitric oxide donor on glucose transport, cGMP content and AMPK activity in human skeletal muscle. Skeletal muscle strips from seven healthy men were incubated in the absence (basal) or presence of spermine NONOate (Spe-NO, nitric oxide donor; 5 mmol/l) or insulin (120 nmol/l). Glucose transport (a), intracellular cyclic GMP (cGMP) content (b), and both AMPK-α1 (c) and AMPK-α2 (d) isoform-specific activity were determined. e AMPK-α1 and AMPK-α2 (f) isoform-specific activity in skeletal muscle from healthy men (n = 2) in response to acute exercise. Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01 vs basal

Fig. 2

Effect of spermine nitric oxide donor on intracellular signalling. Skeletal muscle strips from seven healthy participants were incubated in the absence (basal) or presence of spermine NONOate (Spe-NO, nitric oxide donor; 5 mmol/l) or insulin (120 nmol/l). a Representative immunoblots of protein phosphorylation and abundance. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Bar graphs show quantification for (b) pAkt (Ser⁴⁷³), (c) pTBC1D1/D4, (d) pGSK3α/β (Ser^21/9) and (e) pCaMK II (Thr²⁸⁶). Results (b–e) are mean ± SEM arbitrary units. **p < 0.01 vs basal

Fig. 3

Effect of spermine NONOate and insulin on glucose metabolism and intracellular signalling in L6 myotubes. Myotubes were treated with spermine NONOate (Spe-NO, nitric oxide donor; 20 µmol/l), in the absence (white bars) or presence (black bars) of insulin. a Glucose incorporation into glycogen (n = 13–15) and (b) glucose uptake (n = 10–15) were determined. Results are expressed as mean ± SEM percentage of basal values. Signal transduction was also determined for (c) pAMPK (Thr¹⁷²), (d) pAkt (Ser⁴⁷³), (e) pACC (Ser²²⁷) and (f) pTBC1D1/D4 (n = 5–7). Results are mean ± SEM arbitrary units. *p < 0.05, **p < 0.01 (two-way ANOVA, followed by Bonferroni post-test); ***p < 0.001 (one-way ANOVA, followed by Bonferroni post hoc test); ^† p < 0.0001 vs basal

Fig. 4

Effect of the guanylate cyclase inhibitor LY-83583 on spermine NONOate induced-glycogen synthesis and AMPK phosphorylation. L6 myotubes were treated with spermine NONOate (Spe-NO, nitric oxide donor; 20 µmol/l) and incubated in the absence or presence of LY-835883 (10 µmol/l). Glycogen synthesis (n = 5–8) (a) and AMPK Thr¹⁷² phosphorylation (n = 5–6) (b) were determined. Results are reported as mean ± SEM. Glucose incorporation into glycogen is expressed as percentage of basal. c Protein content of the AMPK-α1 and AMPK-α2 subunits was determined in L6 myotubes. ***p < 0.001 vs basal