Effects of calcineurin activation on insulin-, AICAR- and contraction-induced glucose transport in skeletal muscle

Abstract

Skeletal muscle is composed of fast- and slow-twitch fibres with distinctive physiological and metabolic properties. The calmodulin-activated serine/threonine protein phosphatase calcineurin activates fast- to slow-twitch skeletal muscle remodelling through the induction of the slow-twitch skeletal muscle fibre gene expression programme, thereby enhancing insulin-stimulated glucose uptake and offering protection against dietary-induced insulin resistance. Given the profound influence of skeletal muscle fibre type on insulin-mediated responses, we determined whether the fast- to slow-twitch fibre-type transformation leads to alterations in insulin-independent glucose uptake in transgenic mice expressing a constitutively active form of calcineurin (MCK-CnA* mice). We determined whether skeletal muscle remodelling by activated calcineurin alters glucose transport in response to the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-beta-D-ribofuranoside (AICAR) or muscle contraction, two divergent insulin-independent activators of glucose transport. While insulin-stimulated glucose transport was increased 52%, the AICAR effect on glucose transport was 27% lower in MCK-CnA* mice versus wild-type mice (P < 0.05). In contrast, glucose transport was similar between genotypes after in vitro muscle contraction. Fibre-type transformation was associated with increased AMPKgamma1, decreased AMPKgamma3 and unchanged AMPKgamma2 protein expression between MCK-CnA* and wild-type mice (P < 0.05). The loss of AICAR-mediated glucose uptake is coupled to changes in the AMPK isoform expression, suggesting fibre-type dependence of the AICAR responses on glucose uptake. In conclusion, improvements in skeletal muscle glucose transport in response to calcineurin-induced muscle remodelling are limited to insulin action.

Figure 1. 2-Deoxyglucose uptake

EDL muscles from wild-type or MCK-CnA* mice were incubated under basal conditions (open columns) or stimulated (filled columns) with either insulin (A), AICAR (B), or in vitro muscle contraction (C). Values represent means ± s.e.m. for 4–8 muscles. *P < 0.05, **P < 0.005 compared to wild-type.

Figure 2. Phosphorylation of AMPK

EDL muscles from wild-type or MCK-CnA* mice were incubated under basal conditions (open columns) or stimulated (filled columns) with AICAR (A) or in vitro muscle contraction (B). Values represent means ± s.e.m. for 4–13 muscles. **P < 0.005 compared to wild-type.

Figure 3. Phosphorylation of ACC

EDL muscles from wild-type or MCK-CnA* mice were incubated under basal conditions (open columns) or stimulated (filled columns) with AICAR. Values represent means ± s.e.m. for 8–9 muscles.

Figure 4. AMPKα isoform expression

Protein expression of AMPKα1 and AMPKα2 was determined in EDL muscles from wild-type (open columns) or MCK-CnA* (filled columns) mice by Western blot analysis. Values represent means ± s.e.m. for 8 mice. **P < 0.005 compared to wild-type.

Figure 5. AMPKγ isoform expression

EDL muscles from wild-type (open columns) or MCK-CnA* (filled columns) mice were studied. A, protein expression of AMPKγ1, AMPKγ2 and AMPKγ3 was determined by Western blot analysis. Values represent means ± s.e.m. for 5–8 mice. B, mRNA expression of AMPKγ1, AMPKγ2 and AMPKγ3 was determined by quantitative real-time PCR. Values represent means ± s.e.m. for 9 mice. *P < 0.05, **P < 0.005 compared to wild-type.