Multiple signalling pathways redundantly control glucose transporter GLUT4 gene transcription in skeletal muscle

Abstract

Increased glucose transporter GLUT4 expression in skeletal muscle is an important benefit of regular exercise, resulting in improved insulin sensitivity and glucose tolerance. The Ca(2+)-calmodulin-dependent kinase II (CaMKII), calcineurin and AMPK pathways have been implicated in GLUT4 gene regulation based on pharmacological evidence. Here, we have used a more specific genetic approach to establish the relative role of the three pathways in fast and slow muscles. Plasmids coding for protein inhibitors of CaMKII or calcineurin were co-transfected in vivo with a GLUT4 enhancer-reporter construct either in normal mice or in mice expressing a kinase dead (KD) AMPK mutant. GLUT4 reporter activity was not inhibited in the slow soleus muscle by blocking either CaMKII or calcineurin alone, but was inhibited by blocking both pathways. GLUT4 reporter activity was likewise unchanged in the soleus of KD-AMPK mice, but was significantly reduced by incapacitation of either CaMKII or calcineurin in these mice. On the other hand, in the fast tibialis anterior (TA) muscle, calcineurin appears to exert a prominent role in the control of GLUT4 reporter activity, independent of CaMKII and AMPK. The results point to a muscle type-specific and redundant regulation of GLUT4 enhancer based on the interplay of multiple signalling pathways, all of which are known to affect myocyte enhancing factor 2 (MEF2) transcriptional activity, a point of convergence of different pathways on muscle gene regulation.

Figure 2. CaMKII inhibitor KIIN has no effect on GLUT4 enhancer activity in transfected adult skeletal muscles

Adult mouse soleus and tibialis anterior (TA) muscles were transfected with either KIIN or an empty vector together with the GLUT4 enhancer upstream of luciferase. Luciferase activity is expressed as a percentage of that measured in muscles injected with empty vector alone. Data are mean ±s.e.m. (A, n= 23; B, n= 24).

Figure 1. Validation in vivo of KIIN as inhibitor of CaMKII and of Cain as inhibitor of calcineurin

The activity of a CREB reporter, reflecting the activity of the transcription factor CREB, a well-known target of CaMKII, is inhibited by co-transfection with the plasmid coding for KIIN (A). The activity of an NFAT reporter, reflecting the activity of the transcription factor NFAT, a major target of calcineurin, is inhibited by co-transfection with the plasmid coding for Cain (B). Data are mean ±s.e.m. (n= 4 in A; n= 5 in B). *^,**Significantly different from control vector group (P < 0.05 and 0.01 respectively).

Figure 3. The calcineurin inhibitor Cain inhibits GLUT4 enhancer activity in soleus but not in TA

GLUT4 reporter activity is not affected by cotransfection with Cain in soleus muscle (A) but is significantly inhibited in TA (B). Luciferase activity is expressed as a percentage of that measured in muscles injected with empty vector alone. Data are mean ±s.e.m. (A, n= 20; B, n= 21). **Significantly different from control vector group (P < 0.01).

Figure 4. Simultaneous block of CaMKII and calcineurin inhibits GLUT4 enhancer activity both in soleus and TA

The two signalling pathways were inhibited by co-injection of KIIN and Cain together with GLUT4 enhancer in soleus (A) and TA (B). Luciferase activity is expressed as a percentage of that measured in muscles injected with empty vector alone. Data are mean ±s.e.m. (n= 13 in A and B). **Significantly different from control vector group (P < 0.01).

Figure 5. Transgenic mice with dominant negative AMPK (KD-AMPK) have levels of GLUT4 enhancer activity indistinguishable from wild-type littermates

GLUT4 enhancer activity was compared in adult soleus (A) and TA (B) muscles from KD-AMPK mice and wt littermates, transfected with an empty vector. Luciferase activity is expressed as a percentage of that measured in wt muscles. Data are mean ±s.e.m. (n= 9 in A and 8 in B).

Figure 6. Effect of calcineurin and CaMKII inhibition on GLUT4 enhancer activity in KD-AMPK mice

The effects of KIIN (A and B), Cain (C and D) and of the two inhibitors combined (E and F) were assessed in KD-AMPK mice. Soleus (A, C and E) and TA (B, D and F) were transfected and analysed for luciferase expression. Luciferase activity is expressed as a percentage of that measured in muscles injected with empty vector alone. Data are mean ±s.e.m. (n= 14 for KIIN, 8 for Cain and 5 for KIIN + Cain). * and **, significantly different from control vector group (P < 0.05 and 0.01, respectively).

Figure 7. Scheme of the regulation of GLUT4 transcription in skeletal muscle by the calcineurin (Cn), CaMKII and AMPK pathways via MEF2

The ‘=’ symbol between HDAC4 and HDAC5 indicates the possibility of heterodimerization between the two HDACs (see text for details).